Neisseria spp. polypeptide, nucleic acid sequence and uses thereof

ABSTRACT

The invention discloses the Neisseria spp. NGSP polypeptide, polypeptides derived therefrom (NGSP-derived polypeptides), nucleotide sequences encoding said polypeptides, and antibodies that specifically bind the NGSP polypeptide and/or NGSP-derived polypeptides. Also disclosed are prophylactic or therapeutic compositions, including antigenic, preferably immunogenic compositions, e.g., vaccines, comprising NGSP polypeptide and/or a NGSP-derived polypeptide or antibodies thereto. The invention additionally discloses methods of inducing an immune response to Neisseria and Neisseria NGSP polypeptide and an NGSP-derived polypeptide in animals.

[0001] This application claims priority benefits of provisional U.S.application No. 60/098,685, filed Sep. 1, 1998, the entire disclosure ofwhich is incorporated by reference herein.

INTRODUCTION

[0002] The present invention relates generally to a polypeptide ofNeisseria spp., e.g., of N. gonorrhoeae, of approximately 40-55 kDreferred to as “NGSP”. The invention encompasses an isolated or purifiedNGSP polypeptide, fragments thereof and polypeptides derived therefrom(NGSP-derived polypeptides), and methods of making thereof. Theinvention also encompasses antibodies, including cytotoxic orbactericidal antibodies, that specifically bind the NGSP polypeptide,NGSP-derived polypeptides and/or fragments thereof. The inventionfurther encompasses immunogenic, prophylactic or therapeuticcompositions, including vaccines, that comprise NGSP polypeptide,NGSP-derived polypeptides and/or fragments thereof. The inventionadditionally provides methods of inducing an immune response toNeisseria gonorrhoeae in an animal and methods of treating infections inan animal caused by Neisseria gonorrhoeae. The invention furtherprovides isolated nucleotide sequences encoding the NGSP polypeptide,NGSP-derived polypeptides and fragments thereof, vectors having saidsequences, and host cells containing said vectors.

BACKGROUND OF THE INVENTION

[0003] Neisseriae are gram-negative diplococci and include but are notlimited to Neisseria ovis, Neisseria lacunata, Neisseria osloensis,Neisseria bovis, Neisseria meningitidis, and Neisseria gonorrhoeae andcause a wide range of infectious diseases.

[0004] Although intensely investigated at both the clinical and basicresearch levels for several decades, Neisseria gonorrhoeae remains asexually transmitted disease (STD) of significant health and economicimportance for both the developed as well as the developing world.N.gonorrhoeae is the second most common STD in the United States withover 200,000 cases being reported to the Centers for Disease Control andPrevention (CDC) in 1998. Gonococcal infections are usually localized tothe mucosal surfaces initially contacted by the pathogen such as thecervix, vagina, urethra, conjunctiva, anorectal area, or the pharyngealsurface and where they can produce a variety of clinical diseases suchas cervicitis, urethritis, and conjunctivitis (ophthalmia neonatorum).Acute N.gonorrhoeae infections generally elicit classical symptoms andare often accompanied by a purulent lymphocytic discharge at the site ofinfection. Acute N.gonorrhoeae infections can often be treatedsuccessfully with appropriate antibiotics many gonococcal infections, inboth men and women, remain asymptomatic and can persist for years. Whilemost gonococcal infections remain localized, in some individuals, forreasons yet to be understood, the infection can spread from the primarysite of infection and produce a severe disseminated disease such aspelvic inflammatory disease (PID), bacteremia, and arthritis. Besidescausing adverse consequences to the urogenital tract, infection withN.gonorrhoeae has been shown to facilitate the transmission of the humanimmunodeficiency virus (HIV).

[0005] Fluoroquinolones and broad-spectrum cephalosporins are the mosteffective antimicrobial agents for the treatment of gonorrhea. However,clinically significant resistance to fluoroquinolones has emerged inNeisseria gonorrhoeae and raised concerns regarding the futureeffectiveness and expense of anti-gonococcal treatment regimens. Thedevelopment of an effective vaccine that would prevent gonococcalinfection and/or significantly reduce the sequelae associated withurogenital infection, especially PID, would provide an attractive andproactive method for combating gonococcal infections. Recent advances inour understanding of gonococcal pathogenesis have provided a foundationfor identifying and evaluating antigens, individually and incombination, as potential gonococcal vaccines.

[0006] The HtrA protein has been identified as a virulence factor forseveral bacterial pathogens including, Yersinia enterocolitica, Brucellaabortus, and Salmonella typhimurium. In some but not all organisms HtrAappears to be a stress-responsive protein, possibly contributing to theorganisms' survival under oxidative challenge and/or at elevatedtemperatures. The exact role HtrA plays during the pathogenesis processhas not yet been fully defined. Bacteria-host cell interaction and theresulting signal transduction events that are triggered in the pathogenmay promote expression of the HtrA protein. The E. coli and H.influenzae HtrA proteins, including the Hin47 protein described in U.S.Pat. Nos. 5,679,547 and 5,721,115, both of which are incorporated hereinby reference in their entireties, have been shown to be serine proteasesand possess three relatively conserved domains that house the catalyticresidues H, D and S.

[0007] HtrA is a virulence factor, having serine protease activity,which has recently been identified as a target for the development ofanti-bacterial agents against gram negative bacterial pathogens. (Jonesand Hruby, 1998, New targets for antibiotic development: biogenesis ofsurface adherence structures, DDT Vol. 3(11):495-504; Barrett and Hock,1998, Two-component signal transduction as a target for microbialanti-infective therapy, Antimicrobial. Agents and Chemother.42(7):1529-1536; Fabret and Hock, 1998, A two-component signaltransduction system essential for growth of Bacillus subtillis:implications for anti-infective therapy, J. Bacteriol.,180(23):6375-6382).

[0008] Citation or identification of any reference in this section orany other section of this application shall not be construed as anindication that such reference is available as prior art to the presentinvention.

3. SUMMARY OF THE INVENTION

[0009] One object of this invention is to identify and provide a noveland highly conserved protein (referred to hereafter and in the claims as“NGSP”) from Neisseria spp., preferably Neisseria gonorrhoeae ,Neisseria ovis, Neisseria lacunata, Neisseria osloensis, and Neisseriabovis. The protein of the present invention has a molecular weight ofapproximately 40-55 kD, and has limited similarity (˜36% identityoverall) to the DegP (HtrA) protein of E. coli [% identity determinedusing TBLASTN program (Altschul et al., 1990, J. Molec. Biol.215:403-10; Altschul et al., 1997, Nuc. Acids Res. 25:3389-3402) withdata entered using FASTA format; expect 10 filter default; description100, alignment as described www.ncbi.nlm.nih.gov.] and has not beenpreviously identified in any Neisseria spp. The protein sequence whichis another object of this invention has similarity to severalDegP/HtrA-like serine proteases from two other bacteria and thesesequence homologies have not been previously reported for any Neisseriaspp.

[0010] The invention is based, in part, on the surprising discovery thatNeisseria gonorrhoeae, and various strains and cultivars thereof, have aprotein, NGSP polypeptide, which is about 40 kD to about 55 kD inmolecular weight, preferably about 44 kD to about 53 kD.

[0011] The present invention encompasses the NGSP polypeptide ofNeisseria gonorrhoeae and other Neisseria spp, including but not limitedto, Neisseria ovis, Neisseria lacunata, Neisseria osloensis, andNeisseria bovis, having a molecular weight, as determined from thededuced amino acid sequence, of 40 kD to about 55 kD, in isolated orrecombinant form. A homologous protein, NMASP, from Neisseriameningitidis, is described and claimed in copending application ofApplicants entitled “Neisseria meningitidis Polypeptide, Gene SequenceAnd Uses Thereof” [Attorney Docket No. 7969-083] filed on even dateherewith, which is hereby incorporated by reference in its entirety. Thepresent invention encompasses a purified NGSP polypeptide, polypeptidesincluding fragments, derived therefrom (NGSP-derived polypeptides), andmethods for making said polypeptide and derived polypeptides. Theinvention also encompasses antisera and antibodies, including cytotoxicor bactericidal antibodies, which bind to and are specific for the NGSPpolypeptide, NGSP-derived polypeptides and/or fragments thereof.

[0012] The invention further encompasses pharmaceutical compositionsincluding prophylactic or therapeutic compositions and which may beantigenic, preferably immunogenic compositions including vaccines,comprising one or more of said polypeptides, optionally in combinationwith, fused to or conjugated to one or more other component(s),including a lipid, phospholipid, a carbohydrate including alipopolysaccharide, any protein(s) novel, or known to those skilled inthe art, inactivated whole or attenuated organisms, including but notlimited to any Neisseria, Chlamydia, Moraxella, Pseudomonas,Streptococcus or Haemophilus bacteria. The invention further encompassespharmaceutical compositions including prophylactic or therapeuticcompositions, which may be immunogenic compositions including vaccines,comprising one or more of the NGSP polypeptide and NGSP-derivedpolypeptides and an attenuated or inactivated Neisseria cultivar or anattenuated or inactivated Neisseria cultivar expressing NGSP polypeptidein a greater amount when compared to wild-type Neisseria.

[0013] The invention additionally provides methods of inducing an immuneresponse to Neisseria spp. in an animal, and methods of treating orpreventing an infection caused by Neisseria spp. in an animal. Preferredis N. gonorrhoeae.

[0014] The invention further provides isolated nucleotide sequenceencoding the NGSP polypeptide, NGSP-derived polypeptides, and fragmentsthereof, vectors having said sequences, host cells containing saidvectors, recombinant polypeptides produced therefrom, and pharmaceuticalcompositions comprising the nucleotide sequences, vectors, or cells.

[0015] In other embodiments of the invention there are provided methodsfor identifying compounds which bind to or otherwise interact with andinhibit or activate an activity of a NGSP peptide or polypeptide or theDNA sequences encoding same of the invention comprising: contacting theDNA or polypeptide to assess the binding or other interaction, suchbinding or interaction being associated with a binding or interaction ofthe DNA or polypeptide with the compound and determining whether thecompounds binds to or otherwise interacts with and activates or inhibitsan activity of the DNA or polypeptide by detecting the presence orabsence of a signal generated from the binding or interaction of thecompounds with the DNA or polypeptide. In accordance with another aspectof the invention, there are provided NGSP agonist or antagonists,preferably bacteriostatic acteriocidal agonists or antagonists.

[0016] One advantage of this invention is that antibody generatedagainst the newly iscovered NGSP polypeptide of the present invention,in an animal host will exhibit bactericidal and/or opsonic activityagainst many Neisseriae strains and thus confer broad cross-strainprotection. Bactericidal and/or opsonic antibody will prevent thebacterium from infecting the host and/or enhance the clearance of thepathogen by the host's immune system. Neisseria antibody bactericidalactivity is the principal laboratory test that has been correlated withprotection in humans and is the standard assay in the field as beingpredictive of a vaccine's efficacy against Neisseria infections.Bactericidal antibodies are particularly important for N. gonorrhoeaevaccines because there is no natural animal host other than humans andthus there is no relevant predictive animal model of disease.

3.1. DEFINITIONS AND ABBREVIATIONS

[0017] anti-NGSP = a polyclonal or monoclonal antibody or antiserum thatbinds specifically to a NGSP polypeptide or NGSP-derived polypeptideATCC = American Type Culture Collection blebs naturally occurring outermembrane vesicles of Neisseria antigenic = capable of bindingspecifically to antibody or T cell receptors and provoking an immuneresponse immunogenic capable of provoking a protective cellular orhumoral immune response kD = kilodaltons N. = Neisseria spp. andincludes but is not limited to, Neisseria ovis, Neisseria lacunata,Neisseria osloensis, Neisseria bovis, and Neisseria gonorrhoeae NGSP = anon-cytosolic polypeptide of a Neisseria spp. particularly N.gonorrhoeae, or any strain or cultivar thereof, having a molecularweight of about 40 kD to 55 kD; NGSP-derived fragment of the NGSPpolypeptide; variant of polypeptide = wild-type NGSP polypeptide orfragment thereof, containing one or more amino acid deletions,insertions or substitutions; or chimeric protein comprising aheterologous polypeptide fused to a C-terminal or N-terminal or internalsegment of a whole or a portion of the NGSP polypeptide; OG =n-octyl-β-D-glucopyranoside or octylglucoside PBS = phosphate bufferedsaline PAG = polyacrylamide gel polypeptide = a peptide or protein ofany length, preferably one having eight or more amino acid residues SDS= sodium dodecylsulfate SDS-PAGE = sodium dodecylsulfate polyacrylamidegel electrophoresis

[0018] Nucleotide or nucleic acid sequences defined herein arerepresented by one-letter symbols for the bases as follows:

[0019] A (adenine)

[0020] C (cytosine)

[0021] G (guanine)

[0022] T (thymine)

[0023] U (uracil)

[0024] M (A or C)

[0025] R (A or G)

[0026] W (A or T/U)

[0027] S (C or G)

[0028] Y (C or T/U)

[0029] K (G or T/U)

[0030] V (A or C or G; not T/U)

[0031] H (A or C or T/U; not G)

[0032] D (A or G or T/U; not C)

[0033] B (C or G or T/U; not A)

[0034] N (A or C or G or T/U) or (unknown)

[0035] Peptide and polypeptide sequences defined herein are representedby one-letter or three symbols for amino acid residues as follows: 1letter 3 letter amino acid A Ala (alanine) R Arg (arginine) N Asn(asparagine) D Asp (aspartic acid) C Cys (cysteine) Q Gln (glutamine) EGlu (glutamic acid) G Gly (glycine) H His (histidine) I Ile (isoleucine)L Leu (leucine) K Lys (lysine) M Met (methionine) F Phe (phenylalanine)P Pro (proline) S Ser (serine) T Thr (threonine) W Trp (tryptophan) YTyr (tyrosine) V Val (valine) X Xaa (unknown)

[0036] The present invention may be more fully understood by referenceto the following detailed description of the invention, non-limitingexamples of specific embodiments of the invention and the appendedfigures.

4. BRIEF DESCRIPTION OF THE FIGURES

[0037]FIG. 1: Map of NGSP vector pTLZ-NgHtrA#2.

[0038]FIG. 2: Western blot of NGSP protein (lane 2) expressed from IPTGinduced JM109 (pTLZ-NgHtrA#2). A monoclonal anti-(His)₅ antibodyconjugated to HRP (QiaGen) was used to identify the protein andvisualization of the antibody reactive pattern was achieved on Hyperfilmusing the Amersham ECL chemiluminescence system. Lane 1 shows NovexMultiMark molecular weight markers of glutamic dehydrogenase (62 kD),carbonic anhydrase (42 kD), myoglobin-blue (30 kD), myoglobin-red (22kD), and lysozyme (17 kD).

5. DETAILED DESCRIPTION OF THE INVENTION 5.1. NGSP Polypeptide

[0039] The invention provides an isolated or a substantially pure native(wild type) or recombinantly produced polypeptide, referred to as NGSP,of Neisseria spp. including but is not limited to, Neisseria ovis,Neisseria lacunata, Neisseria osloensis, Neisseria bovis, and Neisseriagonorrhoeae , and various strains or cultivars thereof, but not ofNeisseria meningitidis. The NGSP polypeptide comprises the whole or asubunit of a non-cytosolic protein embedded in, or located in thebacterial envelope, which may include the inner membrane, outer surface,and periplasmic space. The NGSP polypeptide has an apparent molecularweight, as determined from the deduced amino acid sequence, of about 40kD to about 55 kD, preferably about 44 kD to about 53 kD.

[0040] NGSP polypeptide may also be identified as the polypeptide inhydrophobic (salt) or detergent extracts of Neisseria blebs or intactcells that has an apparent molecular weight about 40 kD to about 55 kD,preferably about 44 kD to about 53 kD, as determined by denaturing gelelectrophoresis in 12% PAG with SDS, using formulations as described inHarlow and Lane (Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., Appendix I, 1988).

[0041] In particular embodiments, the NGSP polypeptide is thatobtaihable from any of Neisseria gonorrhoeae, Neisseria ovis, Neisserialacunata, Neisseria osloensis, and Neisseria bovis. Preferred speciesare Neisseria gonorrhoeae. Strains from any of these organisms may beobtained worldwide from any biologicals depository, particularly strainsof Ng. ATCC 9793, ATCC 9827, ATCC 9828, ATCC 9830, ATCC 10150, ATCC10874, ATCC 11688, ATCC 11689, ATCC 19088, ATCC 19424, ATCC 21823, ATCC21824, ATCC 21825, ATCC 23050, ATCC 23051, ATCC 27628, ATCC 27629, ATCC27630, ATCC 27631, ATCC 27632, ATCC 27633, ATCC 31148, ATCC 31149, ATCC31150, ATCC 31151, ATCC 31356, ATCC 31397, ATCC 31398, ATCC 31399, ATCC31400, ATCC 31401, ATCC 31402, ATCC 31403, ATCC 31404, ATCC 31405, ATCC31406, ATCC 31407, ATCC 31426, ATCC 31953, ATCC 33084, ATCC 35201, ATCC35541, ATCC 35542, ATCC 43069, ATCC 43070, ATCC 43785, ATCC 49226, ATCC49498, ATCC 49759, ATCC 49926, ATCC 49981, ATCC 51109, ATCC 51803, ATCC51804, ATCC 53420, ATCC 53421, ATCC 53422, ATCC 53423, ATCC 53424, andATCC 53425.

[0042] In a particular embodiment, the NGSP polypeptide comprises adeduced amino acid sequence as depicted in SEQ ID NO: 4. Particularlypreferred fragments of NGSP have deduced amino acid sequences depictedin SEQ ID NOs: 6 and 8. In another particular embodiment, the NGSPpolypeptide is encoded by the nucleotide sequence of SEQ ID NOs: 3 or 5,with particularly preferred fragments encoded by nucleotide sequencesdepicted in SEQ ID NOs: 1, 2 and 7. In another embodiment, the NGSPpolypeptide comprises an amino acid sequence which is substantiallyhomologous to the deduced amino acid sequence of SEQ ID NO: 4 or aportion thereof or is encoded by a nucleotide sequence substantiallyhomologous to the nucleotide sequence of SEQ ID No: 3 or 5 or a portionthereof.

[0043] As used herein a “substantially homologous” sequence is at least70%, preferably greater than 80%, more preferably greater than 90%identical to a reference sequence of identical size or when compared toa reference sequence when the alignment or comparison is conducted by acomputer homology program or search algorithm known in the art. By wayof example and not limitation, useful computer homology programs includethe following: Basic Local Alignment Search Tool (BLAST)(www.ncbi.nlm.nih.gov) (Altschul et al., 1990, J. of Molec. Biol.,215:403-410, “The BLAST Algorithm; Altschul et al., 1997, Nuc. AcidsRes. 25:3389-3402) a heuristic search algorithm tailored to searchingfor sequence similarity which ascribes significance using thestatistical methods of Karlin and Altschul 1990, Proc. Nat'l Acad. Sci.USA, 87:2264-68; 1993, Proc. Nat'l Acad. Sci. USA 90:5873-77. Fivespecific BLAST programs perform the following tasks:

[0044] 1) The BLASTP program compares an amino acid query sequenceagainst a protein sequence database.

[0045] 2) The BLASTN program compares a nucleotide query sequenceagainst a nucleotide sequence database.

[0046] 3) The BLASTX program compares the six-frame conceptualtranslation products of a nucleotide query sequence (both strands)against a protein sequence database.

[0047] 4) The TBLASTN program compares a protein query sequence againsta nucleotide sequence database translated in all six reading frames(both strands).

[0048] 5) The TBLASTX program compares the six-frame translations of anucleotide query sequence against the six-frame translations of anucleotide sequence database.

[0049] Smith-Waterman (database: European Bioinformatics Institutewwwz.ebi.ac.ukibic_sw/) (Smith-Waterman, 1981, J. of Molec. Biol.,147:195-197) is a mathematically rigorous algorithm for sequencealignments.

[0050] FASTA (see Pearson et al., 1988, Proc. Nat'l Acad. Sci. USA,85:2444-2448) is a heuristic approximation to the Smith-Watermanalgorithm. For a general discussion of the procedure and benefits of theBLAST, Smith-Waterman and FASTA algorithms see Nicholas et al., 1998, “ATutorial on Searching Sequence Databases and Sequence Scoring Methods”(www.psc.edu) and references cited therein.

[0051] By further way of example and not limitation, useful computerhomology algorithms and parameters for determining percent identityinclude the following:

[0052] To determine the percent identity of two amino acid sequences orof two nucleic acids, e.g. between Thy-1 sequences and other knownsequences, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In one embodiment, the two sequences are the samelength.

[0053] The determination of percent identity between two sequences canbe accomplished using a mathematical algorithm. A preferred,non-limiting example of a mathematical algorithm utilized for thecomparison of two sequences is the algorithm of Karlin and Altschul,1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin andAltschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul, et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to a nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to a protein molecules of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules (Id.). When utilizingBLAST, Gapped BLAST, and PSI-Blast programs, the default parameters ofthe respective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example ofa mathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, CABIOS (1989). Such an algorithm isincorporated into the ALIGN program (version 2.0) which is part of theCGC sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.Additional algorithms for sequence analysis are known in the art andinclude ADVANCE and ADAM as described in Torellis and Robotti (1994)Comput. Appl. Biosci., 10:3-5; and FASTA described in Pearson and Lipman(1988) Proc. Natl. Acad. Sci. 85:2444-8. Within FASTA, ktup is a controloption that sets the sensitivity and speed of the search. If ktup=2,similar regions in the two sequences being compared are found by lookingat pairs of aligned residues; if ktup=1, single aligned amino acids areexamined. ktup can be set to 2 or 1 for protein sequences, or from 1 to6 for DNA sequences. The default if ktup is not specified is 2 forproteins and 6 for DNA. For a fuirther description of FASTA parameters,see http://bioweb.pasteur.fr/docs/man/man/fasta.1.html#sect2, thecontents of which are incorporated herein by reference.

[0054] Alternatively, protein sequence alignment may be carried outusing the CLUSTAL W algorithm, as described by Higgins et al., 1996,Methods Enzymol. 266:383-402.

[0055] The percent identity between two sequences can be determinedusing techniques similar to those described above, with or withoutallowing gaps. In calculating percent identity, only exact matches arecounted.

[0056] According to various aspects of the invention, the polypeptidesof the invention are characterized by their apparent molecular weightsbased on the polypeptides' migration in SDS-PAGE relative to themigration of known molecular weight markers. While any molecular weightstandards known in the art may be used with the SDS-PAGE, preferredmolecular weight markers comprise at least glutamic dehydrogenase andcarbonic anhydrase. Other markers include bovine serum albumin, chickenovalbumin and bovine carbonic anhydrase. One skilled in the art willappreciate that the polypeptides of the invention may migratedifferently in different types of gel systems (e.g., different buffers;different types and concentrations of gel, crosslinkers or SDS, etc.).One skilled in the art will also appreciate that the polypeptides mayhave different apparent molecular weights due to different molecularweight markers used with the SDS-PAGE. Hence, the molecular weightcharacterization of the polypeptides of the invention is intended to bedirected to cover the same polypeptides on any SDS-PAGE systems and withany molecular weight markers which might indicate sightly differentapparent molecular weights for the polypeptides than those disclosedherein.

5.2. NGSP-Derived Polypeptides

[0057] An NGSP-derived polypeptide of the invention may be a fragment ofthe NGSP polypeptide. Fragments include those polypeptides having 7 ormore amino acids; preferably 8 or more amino acids; more preferably 9 ormore amino acids; and most preferably 10 or more amino acids of the NGSPpolypeptide.

[0058] The intact NGSP polypeptide may contain one or more amino acidresidues that are not necessary to its immunogenicity. It may be thecase, for example, that only the amino acid residues forming aparticular epitope of the NGSP polypeptide are necessary forinmmunogenic activity. Unnecessary amino acid sequences can be removedor modified by techniques well known in the art, i.e., an NGSP-derivedpolypeptide.

[0059] Preferably, the NGSP-derived polypeptides of the invention areantigenic, i.e., binding specifically to an anti-NGSP antibody and morepreferably, the NGSP-derived polypeptides are immunogenic andimmunologically cross-reactive with the NGSP polypeptide, thus beingcapable of eliciting in an animal an immune response to Neisseria. Morepreferably, the NGSP-derived polypeptides of the invention comprisesequences forming one or more epitopes of the native NGSP polypeptide ofNeisseria (i.e., the epitopes of NGSP polypeptide as it exists in intactNeisseria cells). Such preferred NGSP-derived polypeptides can beidentified by their ability to specifically bind antibodies raised tointact Neisseria cells (e.g., antibodies elicited by formaldehyde orglutaraldehyde fixed Neisseria cells; such antibodies are referred toherein as “anti-whole cell” antibodies). For example, polypeptides orpeptides from a limited or complete protease digestion of the NGSPpolypeptide are fractionated using standard methods and.tested for theirability to bind anti-whole cell antibodies. Reactive polypeptidescomprise preferred NGSP-derived polypeptides. They are isolated andtheir amino acid sequences determined by methods known in the art.

[0060] Also preferably, the NGSP-derived polypeptides of the inventioncomprise sequences that form one or more epitopes of native NGSPpolypeptide that mediate bactericidal or opsonizing antibodies. Suchpreferred NGSP-derived polypeptides may be identified by their abilityto generate antibodies that kill Neisseria spp., particularly, N.gonorrhoeae cells. For example, polypeptides from a limited or completeprotease digestion or chemical cleavage of NGSP polypeptide arefractionated using standard methods, injected into animals and theantibodies produced therefrom tested for the ability to interfere withor kill Neisseria cells. Once identified and isolated, the amino acidsequences of such preferred NGSP-derived polypeptides are determinedusing standard sequencing methods. The determined sequence may be usedto enable production of such polypeptides by synthetic chemical and/orgenetic engineering means.

[0061] These preferred NGSP-derived polypeptides also can be identifiedby using anti-whole cell antibodies to screen bacterial librariesexpressing random fragments of Neisseria genomic DNA or clonednucleotide sequences encoding the whole NGSP polypeptide or fragmentsthereof. See, e.g., Sambrook et al., Molecular Cloning, A LaboratoryManual, 2nd ed., Cold Spring Harbor Press, N.Y., Vol. 1, Chapter 12. Thereactive clones are identified and their inserts are isolated andsequenced to determine the amino acid sequences of such preferredNGSP-derived polypeptides.

[0062] By way of example and not limitation, the unwanted amino acidsequences can be removed or modified by limited proteolytic digestionusing enzymes such as trypsin, papain, or related proteolytic enzymes orby chemical cleavage using agents such as cyanogen bromide and followedby fractionation of the digestion or cleavage products.

[0063] An NGSP-derived polypeptide of the invention may also be amodified NGSP polypeptide or fragment thereof (i.e., an NGSP polypeptideor fragment having one or more amino acid substitutions, insertionsand/or deletions of the wild-type NGSP sequence or amino acidschemically modified in vivo or in vitro). Such modifications may enhancethe immunogenicity of the resultant polypeptide product or have noeffect on such activity. The NGSP-derived polypeptides maintain specificbinding activity to anti-NGSP. Modification techniques that may be usedinclude those disclosed in U.S. Pat. No. 4,526,716.

[0064] As an illustrative, non-limiting example, one or more amino acidresidues within the sequence can be substituted by another amino acid ofa similar polarity which acts as a functional equivalent, resulting in asilent alteration. Substitutes for an amino acid within the sequence maybe selected from other members of the class to which the amino acidbelongs. For example, the nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophanand methionine. The polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine. The positivelycharged (basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid.

[0065] An NGSP-derived polypeptide of the invention may also be amolecule comprising a region that is substantially homologous to (e.g.,in various embodiments, at, least 60% or 70% or 80% or 90% or 95%identity over an amino acid sequence of identical size or when comparedto an aligned sequence in which the alignment is performed by a computerhomology program known in the art) or whose encoding nucleic acid iscapable of hybridizing to a coding NGSP sequence, under highlystringent, moderately stringent, or low or nonstringent conditions.

[0066] By way of example and not limitation, useful computer homologyprograms include the following: Basic Local Alignment Search Tool(BLAST) (www.ncbi.nlm.nih.gov) (Altschul et al., 1990, J. of Molec.Biol., 215:403-410, “The BLAST Algorithm; Altschul et al., 1997, Nuc.Acids Res. 25:3389-3402) a heuristic search algorithm tailored tosearching for sequence similarity which ascribes significance using thestatistical methods of Karlin and Altschul (1990, Proc. Nat'l Acad. Sci.USA, 87:2264-68; 1993, Proc. Nat'l Acad. Sci. USA 90:5873-77). Twospecific BLAST programs perform the following tasks:

[0067] 1) The BLASTP program compares an amino acid query sequenceagainst a protein sequence database; and

[0068] 2) The BLASTN program compares a nucleotide query sequenceagainst a nucleotide sequence database; and hence are useful toidentify, respective substantially homologous amino acid and nucleotidesequences.

[0069] Additional algorithms which can be useful are the Smith-Watermanand FASTA algorithms. See supra Section 5.1 for a more detaileddescription description of useful algorithms and parameters fordetermining percent identity of nucleobtide (and/or amino acid)sequences.

[0070] Included within the scopel of the invention are NGSP-derivedpolypeptides which are NGSP polypeptide fragments or other derivativesor analogs which are differentially modified during or aftertranslation, e.g., by glycosylation, acetylation, phosphorylation,amidation, derivatizatidn by known protecting/blocking groups,proteolytic cleavage, linkage to an antibody molecule or other cellularligand, etc. Any of numerous chemical modifications may be carried outby known techniques, including but not limited to specific chemicalcleavage by cyanogen bromide, trypsin, chypotrypsin, papain, V8protease, NaBH₄; acetylation, formylation, oxidation, reduction;metabolic synthesis in the presence of tunicamycin; etc.

[0071] Furthermore, if desired, nonclassical amino acids or chemicalamino acid analogs can be introduced as a substitution or addition intothe NGSP polypeptide sequence. Non-classical amino acids include but arenot limited to the D-isomers of the common amino acids, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu,ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline,sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine,phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids,designer amino acids such as β-methyl amino acids, Cα-methyl aminoacids, Nα-methyl amino acids, and amino acid analogs in general.Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

[0072] An NGSP-derived polypeptide may further be a chimeric polypeptidecomprising one or more heterologous polypeptides, lipids, phospholipidsor lipopolysaccharides of Neisserial origin or of another bacterialorigin, fused to the amino-terminal or carboxyl-terminal or internal ofa complete NGSP polypeptide or a portion of or a fragment thereof.Useful heterologous polypeptides comprising such chimeric polypeptideinclude, but are not limited to, a) pre- and/or pro-sequences thatfacilitate the transport, translocation and/or processing of theNGSP-derived polypeptide in a host cell, b) affinity purificationsequences, and c) any useful immunogenic sequences (e.g., sequencesencoding one or more epitopes of a surface-exposed protein of amicrobial pathogen). One preferred heterologous protein of the chimericpolypeptide of the invention includes Hin47 (see U.S. Pat. Nos.5,679,547 and 5,721,115).

5.3. Isolation and Purification of NGSP Polypeptide and NGSP-DerivedPolypeptieds

[0073] The invention provides isolated NGSP polypeptides andNGSP-derived polypeptides. As used herein, the term “isolated” meansthat the product is significantly free of other biological materialswith which it is naturally associated. That is, for example, an isolatedNGSP polypeptide composition is between about 70% and 94% pure NGSPpolypeptide by weight. Preferably, the NGSP polypeptides andNGSP-derived polypeptides of the invention are purified. As used herein,the term “purified” means that the product is substantially free ofother biological material with which it is naturally associated. Thatis, a purified NGSP polypeptide composition is at least 95% pure NGSPpolypeptide by weight, preferably at least 98% pure NGSP polypeptide byweight, and most preferably at least 99% pure NGSP polypeptide byweight.

[0074] The NGSP polypeptide of the invention may be isolated from aprotein extract including a whole cell extract, of any Neisseria spp.,including, but not limited to, Neisseria ovis, Neisseria lacunata,Neisseria osloensis, Neisseria bovis, and Neisseria gonorrhoeae (N.g).Preferred species are N.g. Strains from any of these organisms may beobtained worldwide from any biologicals depository, particularly strainsof N.g. ATCC 9793, ATCC 9827, ATCC 9828, ATCC 9830, ATCC 10150, ATCC10874, ATCC 11688, ATCC 11689, ATCC 19088, ATCC 19424, ATCC 21823, ATCC21824, ATCC 21825, ATCC 23050, ATCC 23051, ATCC 27628, ATCC 27629, ATCC27630, ATCC 27631, ATCC 27632, ATCC 27633, ATCC 31148, ATCC 31149, ATCC31150, ATCC 31151, ATCC 31356, ATCC 31397, ATCC 31398, ATCC 31399, ATCC31400, ATCC 31401, ATCC 31402, ATCC 31403, ATCC 31404, ATCC 31405, ATCC31406, ATCC 31407, ATCC 31426, ATCC 31953, ATCC 33084, ATCC 35201, ATCC35541, ATCC 35542, ATCC 43069, ATCC 43070, ATCC 43785, ATCC 49226, ATCC49498, ATCC 49759, ATCC 49926, ATCC 49981, ATCC 51109, ATCC 51803, ATCC51804, ATCC 53420, ATCC 53421, ATCC 53422, ATCC 53423, ATCC 53424, andATCC 53425. Another source of the NGSP polypeptide is a proteinpreparation from a gene expression system expressing a sequence encodingNGSP polypeptide or NGSP-derived polypeptides (see Section 5.7., infra).

[0075] The NGSP polypeptide can be isolated and purified from the sourcematerial using any biochemical technique and approach well known tothose skilled in the art. In one approach, Neisseria cellular envelopeis obtained by standard techniques and inner membrane, periplasmic andouter membrane proteins are solubilized using a solubilizing agent suchas a detergent or hypotonic solution. A preferred detergent solution isone containing octyl glucopyranoside (OG), sarkosyl or TRITON X100™(t-octylphenoxypolyethoxyethanol). A preferred solubilizing hypotonicsolution is one containing LiCl. NGSP polypeptide is, in the solubilizedfraction. Cellular debris and insoluble material in the extract areseparated and removed preferably by centrifuging. The polypeptides inthe extract are concentrated, incubated in SDS-containing Laemmli gelsample buffer at 100° C. for 5 minutes and then fractionated byelectrophoresis in a denaturing sodium dodecylsulfate (SDS)polyacrylamide gel (PAG) from about 6 % to about 12 %, with or without areducing agent. See Laemmli, 1970, Nature 227:680-685. The band orfraction identified as NGSP, polypeptide, having an apparent molecularweight of about 40 kD to about 55 kD, as described above, may then beisolated directly from the fraction or gel slice containing the NGSPpolypeptide. In a preferred embodiment, NGSP polypeptide has an apparentmolecular weight of about 44 kD to about 53 kD which could be determinedby comparing its migration distance or rate in a denaturing SDS-PAGErelative to those of bovine serum albumin (66.2 kD) and chickenovalbumin (45 kD).

[0076] Another method of purifying NGSP polypeptide is by affinitychromatography using anti-NGSP antibodies, (see Section 5.5).Preferably, monoclonal anti-NGSP antibodies are used. The antibodies arecovalently linked to agarose gels activated by cyanogen bromide orsuccinamide esters (Affi-Gel, BioRad, Inc.) or by other. methods knownto those skilled in the art. The protein extract is loaded on the top ofthe gel as described above. The contact is for a period of time andunder standard reaction conditions sufficient for NGSP polypeptide tobind to the antibody. Preferably, the solid support is a material usedin a chromatographic column. NGSP polypeptide is then removed from theantibody, thereby permitting the recovery NGSP polypeptide in isolated,or preferably, purified form.

[0077] An NGSP-derived polypeptide of the invention can be produced bychemical and/or enzymatic cleavage or degradation of isolated orpurified NGSP polypeptide. An NGSP-derived polypeptide can also bechemically synthesized based on the known amino acid sequence of NGSPpolypeptide and, in the case of a chimeric polypeptide, the amino acidsequence of the heterologous polypeptide by methods well known in theart. See, for example, Creighton, 1983, Proteins: Structures andMolecular Principles, W. H. Freeman and Co., NY.

[0078] An NGSP-derived polypeptide can also be produced in a geneexpression system expressing a recombinant nucleotide constructcomprising a sequence encoding NGSP-derived polypeptides. The nucleotidesequences encoding polypeptides of the invention may be synthesized,and/or cloned, and expressed according to techniques well known to thoseskilled in the art. See, for example, Sambrook, et al., 1989, MolecularCloning, A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, N.Y.,Chapter 9.

[0079] NGSP-derived polypeptides of the invention can be fractionatedand purified by the application of standard protein purificationtechniques, modified and applied in accordance with the discoveries andteachings described herein. In particular, preferred NGSP-polypeptidesof the invention, those that form an outer-surface or exposed epitope ofthe native NGSP polypeptide may be isolated and purified according tothe affinity procedures disclosed above for the isolation andpurification of NGSP polypeptide (e.g., affinity purification usinganti-NGSP antibodies).

[0080] If desirable, the polypeptides of the invention may be furtherpurified using standard protein or peptide purification techniquesincluding but not limited to electrophoresis, centrifugation, gelfiltration, precipitation, dialysis, chromatography (including ionexchange chromatography, affinity chromatography, immunoadsorbentaffinity chromatography, reverse-phase high performance liquidchromatography, and gel permeation high performance liquidchromatography), isoelectric focusing, and variations and combinationsthereof.

[0081] One or more of these techniques may be employed sequentially in aprocedure designed to isolate and/or purify the NGSP polypeptide or theNMAP-derived polypeptides of the invention according to its/theirphysical or chemical characteristics. These characteristics include thehydrophobicity, charge, binding capability, and molecular weight of theprotein. The various fractions of materials obtained after eachtechnique are tested for their abilities to bind the NGSP receptor orligand, to bind anti-NGSP antibodies or to have serine protease activity(“test” activities). Those fractions showing such activity are thensubjected to the next technique in the sequential procedure, and the newfractions are tested again. The process is repeated until only onefraction having the above described “test” activities remains and thatfraction produces only a single band or entity when subjected topolyacrylamide gel electrophoresis or chromatography.

5.4. NGSP Immunogens and Anti-NGSP Antibodies

[0082] The present invention provides antibodies that specifically bindNGSP polypeptide or NGSP-derived polypeptides. For the production ofsuch antibodies, isolated or preferably, purified preparations of NGSPpolypeptide or NGSP-derived polypeptides are used as antigens in anantigenic composition, more preferably as immunogens in an immunogeniccomposition.

[0083] In an embodiment, the NGSP polypeptide is separated from otherouter membrane or periplasmic proteins present in the extracts ofNeisseria cells or blebs using SDS-PAGE (see Section 5.3. above) and thegel slice containing NGSP polypeptide is used as an immunogen andinjected into a rabbit to produce antisera containing polyclonal NGSPantibodies. The same immunogen can be used to immunize mice for theproduction of hybridoma lines that produce monoclonal anti-NGSPantibodies. In particular embodiments, the immunogen is a PAGE slicecontaining isolated or purified NGSP from any Neisseria gonorrhoeae ,including, but not limited to, Neisseria ovis, Neisseria lacunata,Neisseria osloensis, Neisseria bovis, and Neisseria gonorrhoeae (N.g.).Preferred species are N.g. Particularly preferred are the strains ofN.g. ATCC 9793, ATCC 9827, ATCC 9828, ATCC 9830, ATCC 10150, ATCC 10874,ATCC 11688, ATCC,11689, ATCC 19088, ATCC 19424, ATCC 21823, ATCC 21824,ATCC 21825, ATCC 23050, ATCC 23051, ATCC 27628, ATCC 27629, ATCC 27630,ATCC 27631, ATCC 27632, ATCC 27633, ATCC 31148, ATCC 31149, ATCC 31150,ATCC 31151, ATCC 31356, ATCC 31397, ATCC 31398, ATCC 31399, ATCC 31400,ATCC 31401, ATCC 31402, ATCC 31403, ATCC 31404, ATCC 31405, ATCC 31406,ATCC 31407, ATCC 31426, ATCC 31953, ATCC 33084, ATCC 35201, ATCC 35541,ATCC 35542, ATCC 43069, ATCC 43070, ATCC 43785, ATCC 49226, ATCC 49498,ATCC 49759, ATCC 49926, ATCC 49981, ATCC 51109, ATCC 51803, ATCC 51804,ATCC 53420, ATCC 53421, ATCC 53422, ATCC 53423, ATCC 53424, and ATCC53425.

[0084] In other embodiments, peptide fragments of NGSP polypeptide areused as immunogens. Preferably, peptide fragments of purified NGSPpolypeptide are used. The peptides may be produced by proteasedigestion, chemical cleavage of isolated or purified NGSP polypeptide orchemical synthesis and then may be isolated or purified. Such isolatedor purified peptides can be used directly as immunogens. In particularembodiments, useful peptide fragments are 5 or more amino acids inlength.

[0085] Useful immunogens may also comprise such peptides or peptidefragments conjugated to a carrier molecule, preferably a carrierprotein. Carrier proteins may be any commonly used in immunology,include, but are not limited to, bovine serum albumin (BSA), chickenalbumin, keyhole limpet hemocyanin (KLH) and the like. For a discussionof hapten protein conjugates, see, for example, Hartlow, et al.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1988, or a standard immunology textbook suchas Roitt, I. et al., IMMUNOLOGY, C.V. Mosby Co., St. Louis, Mo. (1985)or Klein, J., IMMUNOLOGY, Blackwell Scientific Publications, Inc.,Cambridge, Mass., (1990).

[0086] In yet another embodiment, for the production of antibodies thatspecifically bind one or more epitopes of the native NGSP polypeptide,intact Neisseria cells or blebs prepared therefrom are used asimmunogen. The cells or blebs may be fixed with agents such asformaldehyde or glutaraldehyde before immunization. See Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1988, Chapter 15. It is preferred that,suchanti-whole cell antibodies be monoclonal antibodies. Hybridoma linesproducing the desired monoclonal antibodies can be identified by usingpurified NGSP polypeptide as the screening ligand. The immunogen forinducing these antibodies are whole cells, blebs, extracts or lysates ofany Neisseria, including, but not limited to, Neisseria ovis, Neisserialacunata, Neisseria osloensis, Neisseria bovis, and Neisseriagonorrhoeae (N.g.). Preferred species are N.g. Particularly preferredare strains of N.g. ATCC 9793, ATCC 9827, ATCC 9828, ATCC 9830, ATCC10150, ATCC 10874, ATCC 11688, ATCC 11689, ATCC 19088, ATCC 19424, ATCC21823, ATCC 21824, ATCC 21825, ATCC 23050, ATCC 23051, ATCC 27628, ATCC27629, ATCC 27630, ATCC 27631, ATCC 27632, ATCC 27633, ATCC 31148, ATCC31149, ATCC 31150, ATCC 31151, ATCC 31356, ATCC 31397, ATCC 31398, ATCC31399, ATCC 31400, ATCC 31401, ATCC 31402, ATCC 31403, ATCC 31404, ATCC31405, ATCC 31406, ATCC 31407, ATCC 31426, ATCC 31953, ATCC 33084, ATCC35201, ATCC 35541, ATCC 35542, ATCC 43069, ATCC 43070, ATCC 43785, ATCC49226, ATCC 49498, ATCC 49759, ATCC 49926, ATCC 49981, ATCC 51109, ATCC51803, ATCC 51804, ATCC 53420, ATCC 53421, ATCC 53422, ATCC 53423, ATCC53424, and ATCC 53425.

[0087] Polyclonal antibodies produced by whole cell or blebimmunizations contain antibodies that bind other Neisseria proteins(“non-anti-NGSP antibodies”) and thus are more cumbersome to use whereit is known or suspected that the sample contains other Neisseriaproteins or materials that are cross-reactive with these other proteins.Under such circumstances, any binding by the anti-whole cell antibodiesof a given sample or band must be verified by coincidental binding ofthe same sample or band by antibodies that specifically bind NGSPpolypeptide (e.g., anti-NGSP) and/or a NGSP-derived polypeptide, or bycompetition tests using anti-NGSP antibodies, NGSP polypeptide orNGSP-derived polypeptide as the competitor (i.e., addition of anti-NGSPantibodies, NGSP polypeptide or NGSP-derived polypeptide to the reactionmix lowers or abolishes sample binding by anti-whole cell antibodies).Alternatively, such polyclonal antisera, containing “non-anti-NGSP”antibodies, may be cleared of such antibodies by standard approaches andmethods. For example, the non-anti-NGSP antibodies may be removed byprecipitation with cells of a NGSP deletion or “knockout” mutantNeisseria cultivars or Neisseria strains known not to have the NGSPpolypeptide; or by absorption to columns comprising such cells or outermembrane proteins of such cells.

[0088] In further embodiments, useful immunogens for elicitingantibodies of the invention comprise mixtures of two or more of any ofthe above-mentioned individual immunogens.

[0089] Immunization of animals with the immunogens described herein,preferably humans, rabbits, rats, mice, sheep, goats, cows or horses, isperformed following procedures well known to those skilled in the art,for purposes of obtaining antisera containing polyclonal antibodies orhybridoma lines secreting monoclonal antibodies.

[0090] Monoclonal antibodies can be prepared by standard techniques,given the teachings contained herein. Such techniques are disclosed, forexample, in U.S. Pat. No. 4,271,145 and U.S. Pat. No. 4,196,265.Briefly, an animal is immunized with the immunogen. Hybridomas areprepared by fusing spleen cells from the immunized animal with myelomacells. The fusion products are screened for those producing antibodiesthat bind to the immunogen. The positive hybridomas clones are isolated,and the monoclonal antibodies are recovered from those clones.

[0091] Immunization regimens for production of both polyclonal andmonoclonal antibodies are well known in the art. The immunogen may beinjected by any of a number of routes, including subcutaneous,intravenous, intraperitoneal, intradermal, intramuscular, mucosal, or acombination of these. The immunogen may be injected in soluble form,aggregate form, attached to a physical carrier, or mixed with anadjuvant, using methods and materials well known in the art. Theantisera and antibodies may be purified using column chromatographymethods well -known to those of skill in the art.

[0092] According to the present invention, NGSP polypeptides ofNeisseria strains are immuno-cross reactive. Thus,.antibodies raised toNGSP polypeptide of one Neisseria species, strain or cultivar,specifically bind NGSP polypeptide and NGSP-derived polypeptides ofother Neisseria species, strains and cultivars. For example, polyclonalanti-NGSP antibodies induced by NGSP polypeptide of N.g. specificallybind not only the identical strain NGSP polypeptide (i.e., the NGSPpolypeptide of N.g.) but also NGSP olypeptide and/or NGSP-derivedpolypeptides of other Neisseria, including, but not limited to,Neisseria ovis, Neisseria lacunata, Neisseria osloensis, Neisseriabovis, Neisseria meningitidis, and Neisseria gonorrhoeae (N.g.).Preferred species are N.g.

[0093] The antibodies of the invention, including but not limited toanti-NGSP antibodies, can be used to facilitate isolation andpurification of NGSP polypeptide and NGSP-derived polypeptides. Theantibodies may also be used as probes for identifying clones inexpression libraries that have inserts encoding NGSP polypeptide orfragments thereof. The antibodies may also be used in immunoassays(e.g., ELISA, RIA, Westerns) to specifically detect and/or quantitateNeisseria in biological specimens. Anti-NGSP antibodies of the inventionspecifically bind NGSP polypeptide from Neisseria, including, but notlimited to, Neisseria ovis, Neisseria lacunata, Neisseria osloensis,Neisseria bovis, Neisseria meningitidis, and Neisseria gonorrhoeae. Thusanti-NGSP antibodies can be used to diagnose Neisseria infections.

[0094] The antibodies of the invention, particularly those which arecytotoxic, may also be used in passive immunization to prevent orattenuate Neisseria infections of animals, including humans. (As usedherein, a cytotoxic antibody is one which enhances opsonization and/orcomplement killing of the bacterium bound by the antibody). An effectiveconcentration of polyclonal or monoclonal antibodies raised against theimmunogens of the invention may be administered to a host to achievesuch effects. The exact concentration of the antibodies administeredwill vary according to each specific antibody preparation, but may bedetermined using standard techniques well known to those of ordinaryskill in the art. Administration of the antibodies may be accomplishedusing a variety of techniques, including, but not limited to thosedescribed in Section 5.6. for the delivery of vaccines.

5.5. Compositions

[0095] The present invention also provides therapeutic and prophylacticcompositions, which may be immunogenic compositions including vaccines,against Neisseria infections of animals, including mammals, and morespecifically rodents and primates, including humans. Preferredimmunogenic compositions include vaccines for use in humans. Theimmunogenic compositions of the present invention can be prepared bytechniques known to those skilled in the art and would comprise, forexample, an immunologically effective amount of any of the NGSPimmunogens disclosed in Section 5.4., optionally in combination with orfused to or conjugated to one or more other immunogens, includinglipids, phospholipids, carbohydrates, lipopolysaccharides, inactivatedor attenuated whole organisms and other proteins, of Neisserial originor other bacterial origin, a pharmaceutically acceptable carrier,optionally an appropriate adjuvant, and optionally other materialstraditionally found in vaccines. Such a cocktail vaccine (comprisingseveral immunogens) has the advantage that immunity against one orseveral pathogens can be obtained by a single administration. Examplesof other immunogens include, but are not limited to, those used in theknown DPT vaccines, entire organisms or subunits therefrom of Chlamydia,HIV, Haemophilus influenzae, Moraxella catarrhalis, and Streptococcuspneumoniae, etc.

[0096] The term “immunologically effective amount” is used herein tomean an amount sufficient to induce an immune response to produceantibodies, in the case of a humoral immune response, and/or cytokinesand other cellular immune response components. Preferably, theimmunogenic composition is one that prevents Neisseria infections orattenuates the severity of many preexisting or subsequent Neisseriainfection. An immunologically effective amount of the immunogen to beused in the vaccine is determined by means known in the art in view ofthe teachings herein. The exact concentration will depend upon thespecific immunogen to be administered, but can be determined by usingstandard techniques, well known to those skilled in the art for assayingthe development of an immune response.

[0097] The combined immunogen and carrier or diluent may be an aqueoussolution, emulsion or suspension or may be a dried preparation. Ingeneral, the quantity of polypeptide immunogen will be between 0.1 and500 micrograms per dose. The carriers are known to those skilled in theart and include stabilizers, diluents, and buffers. Suitable stabilizersinclude carbohydrates, such as sorbitol, lactose, mannitol, starch,sucrose, dextran, and glucose and proteins, such as albumin or casein.Suitable diluents include saline, Hanks Balanced Salts, and Ringerssolution. Suitable buffers include an alkali metal phosphate, an alkalimetal carbonate, or an alkaline earth metal carbonate.

[0098] The immunogenic compositions, including vaccines, may alsocontain one or more adjuvant or immunostimulatory compounds to improveor enhance the immunological response. Suitable adjuvants include, butare not limited to, peptides including bacterial toxins, such as but notlimited to heat labile toxin and/or verotoxin of E. coli, cholera toxin,and shiga toxin and toxoids and/or attenuated forms thereof, chemokines,cytokines and the like; aluminum hydroxide; aluminum phosphate; aluminumoxide; a composition that consists of a mineral oil, such as Marcol 52,or a vegetable oil, and one or more emulsifying agents or surface activesubstances such as saponins, lysolecithin, polycations, polyanions; andpotentially useful human adjuvants such as BCG, QS21, MPL andCorynebacterium parvum.

[0099] The immunogenic compositions, including vaccines, of theinvention are prepared by techniques known to those skilled in the art,given the teachings contained herein. Generally, an immunogen is mixedwith the carrier to form a solution, suspension, or emulsion. One ormore of the additives discussed above may be in the carrier or may beadded subsequently. The vaccine preparations may be desiccated, forexample, by freeze drying or spray drying for storage or formulationspurposes. They may be subsequently reconstituted into liquid vaccines bythe addition of an appropriate liquid carrier or administered in dryformulation known to those skilled in the art, particularly in capsulesor tablet forms.

[0100] The immunogenic compositions, including vaccines, areadministered to humans or other animals, preferably other mammals, suchas ruminants, rodents and primates. They can be administered in one ormore doses. The vaccines may be administered by known routes ofadministration. Many methods may be used to introduce the vaccineformulations described here. These methods include but are not limitedto oral, intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, and intranasal routes. The preferred routes areintradermal, intramuscular or subcutaneous injection.

[0101] The invention also provides a method for inducing an immuneresponse to Neisseria in an animal to generate a humoral and/or cellularimmune response. The method comprises administering an immunologicallyeffective amount of an immunogen of the invention to a host and,preferably, administering a vaccine of the invention to a host.

5.6. Nucleic Acids Encoding the NGSP Polypeptide and NGSP-DerivedPolypeptides

[0102] The present invention also provides nucleic acids, DNA and RNA,encoding NGSP polypeptide and NGSP-derived polypeptides andpharmaceutical compositions comprising same. In a particular embodiment,the NGSP polypeptide comprises a deduced amino acid sequence as depictedin SEQ ID NOs: 4 or 6 and the nucleic acids comprise nucleotidesequences encoding said amino acid sequences. Particularly preferredfragments of NGSP have 5, 6, 7, or more deduced amino acid from theamino acid sequences depicted in SEQ ID NOs: 6 and 8 or sequencessubstantially homologous thereto and the invention encompasses nucleicacids comprising nucleotides encoding said amino acid sequences. Inanother particular embodiment, the NGSP polypeptide is encoded by thenucleotide sequence of SEQ ID NOs: 3 or 5, with particularly preferredfragments depicted in SEQ ID NOs: 1, 2 and 7 or sequences substantiallyhomologous thereto.

[0103] Nucleic acids of the present invention can be single or doublestranded. The invention also provides nucleic acids hybridizable to orcomplementary to the foregoing sequences. In specific aspects, nucleicacids are provided which comprise a sequence complementary to at least10, 15, 25, 50, 100, 200, or 250 contiguous nucleotides of a nucleicacid encoding NGSP polypeptide or an NGSP-derived polypeptide. In aspecific embodiment, a nucleic acid which is hybridizable to a nucleicacid encoding NGSP polypeptide (e.g., having sequence SEQ ID NO.: 3 or5), or to a nucleic acid encoding an NGSP-derived polypeptide, underconditions of low stringency is provided.

[0104] By way of example and not limitation, procedures using suchconditions of low stringency are as follows (see also Shilo andWeinberg, 1981, Proc. Natl. Acad. Sci. USA 78:6789-6792): Filterscontaining DNA are pretreated for 6 h at 40° C. in a solution containing35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1%Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizationsare carried out in the same solution with the following modifications:0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10%(wt/vol) dextran sulfate, and 5-20×10⁶ cpm ³²P-labeled probe is used.Filters are incubated in hybridization mixture for 18-20 h at 40° C.,and then washed for 1.5 h at 55° C. in a solution containing 2×SSC, 25mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution isreplaced with fresh solution and incubated an additional 1.5 h at 60° C.Filters are blotted dry and exposed for autoradiography. If necessary,filters are washed for a third time at 65-68° C. and re-exposed to film.Other conditions of low stringency which may be used are well known inthe art (e.g., as employed for cross-species hybridizations).

[0105] In another specific embodiment, a nucleic acid which ishybridizable to a nucleic acid encoding NGSP polypeptide or anNGSP-derived polypeptide under conditions of high stringency isprovided. By way of example and not limitation, procedures using suchconditions of high stringency are as follows: Prehybridization offilters containing DNA is carried out for 8 h to overnight at 65° C. inbuffer composed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP,0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA.Filters are hybridized for 48 h at 65° C. in prehybridization mixturecontaining 100 μg/ml denatured salmon sperm DNA and 5-20×10⁶ cpm of³²P-labeled probe. Washing of filters is done at 37° C. for 1 h in asolution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. Thisis followed by a wash in 0.1×SSC at 50° C. for 45 min beforeautoradiography. Other conditions of high stringency which may be usedare well known in the art.

[0106] In another specific embodiment, a nucleic acid which ishybridizable to a nucleic acid encoding NGSP polypeptide or anNGSP-derived polypeptide under conditions of moderate stringency isprovided.

[0107] Various other stringency conditions which promote nucleic acidhybridization can be used. For example, hybridization in 6×SSC at about45° C., followed by washing in 2×SSC at 50° C. may be used.Alternatively, the salt concentration in the wash step can range fromlow stringency of about 5×SSC at 50° C., to moderate stringency of about2×SSC at 50° C., to high stringency of about 0.2×SSC at 50° C. Inaddition, the temperature of the wash step can be increased from lowstringency conditions at room temperature, to moderately stringentconditions at about 42° C., to high stringency conditions at about 65°C. Other conditions include, but are not limited to, hybridizing at 68°C. in 0.5M NaHPO₄ (pH7.2)/1 mM EDTA/7% SDS, or hybridization in 50%formamide/0.25M NaHPO₄ (pH 7.2)/0.25 M NaCl/1 mM EDTA/7% SDS; followedby washing in 40 mM NaHPO₄ (pH 7.2)/1 mM EDTA/5% SDS at 42° C. or in 40mM NaHPO₄ (pH7.2) 1 mM EDTA/1% SDS at 50° C. Both temperature and saltmay be varied, or alternatively, one or the other variable may remainconstant while the other is changed.

[0108] Low, moderate and high stringency conditions are well known tothose of skill in the art, and will vary predictably depending on thebase composition of the particular nucleic acid sequence and on thespecific organism from which the nucleic acid sequence is derived. Forguidance regarding such conditions see, for example, Sambrook et al.,1989, Molecular Cloning, A Laboratory Manual, Second Edition, ColdSpring Harbor Press, N.Y., pp. 9.47-9.57; and Ausubel et al., 1989,Current Protocols in Molecular Biology, Green Publishing Associates andWiley Interscience, N.Y.

[0109] Nucleic acids encoding NMAP-derived polypeptides, including butnot limited to fragments or a portion thereof, (see Section 5.2), andNGSP antisense nucleic acids are additionally provided. As is readilyapparent, as used herein, a “nucleic acid encoding a fragment or portionof a nucleic acid encoding NGSP polypeptide or an NGSP-derivedpolypeptide” shall be construed as referring to a nucleic acid encodingonly the recited fragment or portion of the nucleic acid encoding NGSPpolypeptide or an NGSP-derived polypeptide and not the other contiguousportions of the nucleic acid encoding NGSP polypeptide or anNGSP-derived polypeptide protein as a continuous sequence.

[0110] Also encompassed are nucleotide sequences substantiallyhomologous to the above described nucleic acids. As used herein a“substantially homologous” sequence is at least 70%, preferably greaterthan 80%, more preferably greater than 90% identical to a referencesequence of identical size or when the alignment or comparison isconducted by a computer homology program or search algorithm known inthe art.

[0111] By way of example and not limitation, useful computer homologyprograms include the following: Basic Local Alignment Search Tool(BLAST) (www.ncbi.nlm.nih.gov) (Altschul et al., 1990, J. of Molec.Biol., 215:403-410, “The BLAST Algorithm; Altschul et al., 1997, Nuc.Acids Res. 25:3389-3402) a heuristic search algorithm tailored tosearching for sequence similarity which ascribes significance using thestatistical methods of Karlin and Altschul (1990, Proc. Nat'l Acad. Sci.USA, 87:2264-68; 1993, Proc. Nat'l Acad. Sci. USA 90:5873-77). Fivespecific BLAST programs are provided and the BLASTN program compares anucleotide query sequence against a nucleotide sequence database.Additional algorithms which can be useful are the Smith-Waterman andFASTA algorithms. See supra Section 5.1 for a more detailed descriptionof useful algorithms and parameters for determining percent identity ofnucleotide (and/or amino acid) sequences.

[0112] In one aspect, the nucleic acids of the invention may besynthesized using methods known in the art. Specifically, a portion ofor the entire amino acid sequence of NGSP polypeptide or an NGSP-derivedpolypeptide may be determined using techniques well known to those ofskill in the art;, such as via the Edman degradation technique (see,e.g., Creighton, 1983, Proteins: Structures and Molecular Principles,W.H. Freeman & Co., N.Y., pp.34-49). The amino acid sequence obtained isused as a guide for the synthesis of DNA encoding NGSP polypeptide orNGSP-derived polypeptide using conventional chemical approaches orpolymerase chain reaction (PCR) amplification of overlappingoligonucleotides.

[0113] In another aspect, the amino acid sequence may be used as a guidefor synthesis of oligonucleotide mixtures which in turn can be used toscreen for NGSP polypeptide coding sequences in Neisseria genomiclibraries and PCR amplification products. Preferably the DNA used as thesource of the NGSP polypeptide coding sequence, for both genomiclibraries and PCR amplification, is prepared from cells of anyNeisseria, including, but not limited to, Neisseria ovis, Neisserialacunata, Neisseria osloensis, Neisseria bovis, Neisseria meningitidis(N.m.) particularly including types A-L and W, and Neisseria gonorrhoeae(N.g). Preferred species are N.g. Strains from any of these organismsmay be obtained worldwide from any biologicals depository, particularlystrains of N.g. ATCC 9793, ATCC 9827, ATCC 9828, ATCC 9830, ATCC 10150,ATCC 10874, ATCC 11688, ATCC 11689, ATCC 19088, ATCC 19424, ATCC 21823,ATCC 21824, ATCC 21825, ATCC 23050, ATCC 23051, ATCC 27628, ATCC 27629,ATCC 27630, ATCC 27631, ATCC 27632, ATCC 27633, ATCC 31148, ATCC 31149,ATCC 31150, ATCC 31151, ATCC 31356, ATCC 31397, ATCC 31398, ATCC 31399,ATCC 31400, ATCC 31401, ATCC 31402, ATCC 31403, ATCC 31404, ATCC 31405,ATCC 31406, ATCC 31407, ATCC 31426, ATCC 31953, ATCC 33084, ATCC 35201,ATCC 35541, ATCC 35542, ATCC 43069, ATCC 43070, ATCC 43785, ATCC 49226,ATCC 49498, ATCC 49759, ATCC 49926, ATCC 49981, ATCC 51109, ATCC 51803,ATCC 51804, ATCC 53420, ATCC 53421, ATCC 53422, ATCC 53423, ATCC 53424,and ATCC 53425.

[0114] In the preparation of genomic libraries, DNA fragments aregenerated, some of which will encode parts or the whole of NeisseriaNGSP polypeptide. The DNA may be cleaved at specific sites using variousrestriction enzymes. Alternatively, one may use DNase in the presence ofmanganese to fragment the DNA, or the DNA can be physically sheared, asfor example, by sonication and the like. The DNA fragments can then beseparated according to size by standard techniques, including but notlimited to, agarose and polyacrylamide gel electrophoresis, columnchromatography and sucrose gradient centrifugation. The DNA fragmentscan then be inserted into suitable vectors, including but not limited toplasmids, cosmids, bacteriophages lambda or T₄, and yeast artificialchromosome (YAC). (See, for example, Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.; Glover, D. M. (ed.), 1985, DNA Cloning:A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II.) Thegenomic library may be screened by nucleic acid hybridization to labeledprobe (Benton and Davis, 1977, Science 196:180; Grunstein and Hogness,1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961).

[0115] The genomic libraries may be screened with a labeled degenerateoligonucleotide probe corresponding to the amino acid sequence of anypeptide fragment of the NGSP polypeptide using optimal approaches wellknown in the art. Any probe used preferably is 15 nucleotides or longer.Examples of particular probes are described below.

[0116] Clones in libraries with insert DNA encoding the NGSP polypeptideor fragments thereof will hybridize to one or more of the degenerateoligonucleotide probes. Hybridization of such oligonucleotide probes togenomic libraries are carried out using methods known in the art. Any ofthe hybridization procedures described in detail above in this Sectioncan be used. For a specific illustrative example, hybridization with thetwo above-mentioned oligonucleotide probes may be carried out in 2×SSC,1.0% SDS at 50° C. and washed using the same conditions.

[0117] In yet another aspect, clones of nucleotide sequences encoding apart or the entire NGSP polypeptide or NGSP-derived polypeptides mayalso be obtained by screening Neisseria expression libraries. Forexample, Neisseria DNA is isolated and random fragments are prepared andligated into an expression vector (e.g., a bacteriophage, plasmid,phagemid or cosmid) such that the inserted sequence in the vector iscapable of being expressed by the host cell into which the vector isthen introduced. Various screening assays can then be used to select forthe expressed NGSP polypeptide or NGSP-derived polypeptides. In oneembodiment, the various anti-NGSP antibodies of the invention (seeSection 5.5) can be used to identify the desired clones using methodsknown in the art. See, for example, Harlow and Lane, 1988, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., Appendix IV. Clones or plaques from the library arebrought into contact with the antibodies to identify those clones thatbind.

[0118] In an embodiment, colonies or plaques containing DNA that encodesNGSP polypeptide or NGSP-derived polypeptide could be detected usingDYNA Beads according to Olsvick et al., 29th ICAAC, Houston, Tex. 1989,incorporated herein by reference. Anti-NGSP antibodies are crosslinkedto tosylated DYNA Beads M280, and these antibody-containing beads thenare used to adsorb to colonies or plaques expressing NGSP polypeptide orNGSP-derived polypeptide. Colonies or plaques expressing NGSPpolypeptide or NGSP-derived polypeptide is identified as any of thosethat bind the beads.

[0119] Alternatively, the anti-NGSP antibodies can be nonspecificallyimmobilized to a suitable support, such as protein A or G resins, silicaor Celite™ resin. This material is then used to adsorb to bacterialcolonies expressing NGSP polypeptide or NGSP-derived polypeptide asdescribed in the preceding paragraph.

[0120] In another aspect, PCR amplification may be used to producesubstantially pure DNA encoding a part of or the whole of NGSPpolypeptide from Neisseria genomic DNA. Oligonucleotide primers,degenerate or otherwise, corresponding to NGSP polypeptide sequencespresently taught can be used as primers. In particular embodiments, aconvergent set of oligonucleotides, degenerate or otherwise, specificfor the NGSP coding sequences of SEQ ID NOs: 3 or 5 may be used toproduce NGSP-encoding DNA.

[0121] As examples, an oligonucleotide encoding the N-terminal segmentof the NGSP polypeptide may be used as the 5′ forward primer, andtogether with a 3′ reverse PCR oligonucleotide complementary to aninternal, downstream protein coding sequence may be used to amplify anN-terminal-specific NGSP DNA fragment. Alternatively, an oligonucleotideencoding an internal NGSP coding sequence may be used as the 5′ forwardPCR primer together with a 3′ reverse PCR oligonucleotide complementaryto downstream, internal NGSP protein coding sequences may be used to PCRamplify an internal NGSP-specific DNA fragment. Alternatively theforward primer can be combined together with an oligonucleotidecomplementary to the C-terminal NGSP coding region to PCR amplify theNGSP ORF. These NGSP-specific PCR products can be cloned intoappropriate expression vectors to direct the synthesis of all or part ofthe NGSP polypeptide. Alternatively, these NGSP-specific PCR productscan be appropriately labelled and used as hybridization probes toidentify all or part of the NGSP gene from genomic DNA libraries.

[0122] PCR can be carried out, e.g., by use of a Perkin-Elmer Cetusthermal cycler and Taq polymerase (Gene Amp™). One can choose tosynthesize several different degenerate primers, for use in the PCRreactions. It is also possible to vary the stringency of hybridizationconditions used in priming the PCR reactions, to allow for greater orlesser degrees of nucleotide sequence similarity between the degenerateprimers and the corresponding sequences in Neisseria DNA. Aftersuccessful amplification of a segment of the sequence encoding NGSPpolypeptide, that segment may be molecularly cloned and sequenced, andutilized as a probe to isolate a complete genomic clone. This, in turn,permits the determination of the gene's complete nucleotide sequence,the analysis of its expression, and the production of its proteinproduct for functional analysis, as described infra.

[0123] Once an NGSP polypeptide coding sequence has been isolated fromone Neisseria species, strain or cultivar, it is possible to use thesame approach to isolate NGSP polypeptide coding sequences from otherNeisseria species, strains and cultivars. It will be recognized by thoseskilled in the art that the DNA or RNA sequence encoding NGSPpolypeptide (or fragments thereof) of the invention can be used toobtain other DNA or RNA sequences that hybridize with it underconditions of moderate to high stringency, using general techniquesknown in the art. Hybridization with an NGSP sequence from one Neisseriastrain or.cultivar under high stringency conditions will identify thecorresponding sequence from other strains and cultivars. High stringencyconditions vary with probe length and base composition. The formulae fordetermining such conditions are well known in the art. See Sambrook etal., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, N.Y., Chapter 11. As used herein high stringency hybridizationconditions as applied to probes of greater than 300 bases in lengthinvolve a final wash in 0.1×SSC/0.1% SDS at 68° C. for at least 1 hour(Ausubel, et al., Eds., 1989, Current Protocols in Molecular Biology,Vol. I, Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,New York, at page 2.10.3). In particular embodiments, the highstringency wash in hybridization using a probe, for instance, having thesequence of SEQ ID NO:8 or 9 or its complement, is 2×SSC, 1% SDS at 50°C. for about 20 to about 30 minutes.

[0124] One skilled in the art would be able to identify complete clonesof NGSP polypeptide coding sequence using approaches well known in theart. The extent of NGSP. polypeptide coding sequence contained in anisolated clone may be ascertained by sequencing the cloned insert andcomparing the deduced size of the polypeptide encoded by the openreading frames (ORFs) with that of NGSP polypeptide and/or by comparingthe deduced amino acid sequence with that of known amino acid sequenceof purified NGSP polypeptide. Where a partial clone of NGSP polypeptidecoding sequence has been isolated, complete clones may be isolated byusing the insert of the partial clone as hybridization probe.Alternatively, a complete NGSP polypeptide coding sequence can bereconstructed from overlapping partial clones by splicing their insertstogether.

[0125] Complete clones may be any that have ORFs with deduced amino acidsequence matching or substantially homologous to that of NGSPpolypeptide or, where the complete amino acid sequence of the latter isnot available, that of a peptide fragment of NGSP polypeptide and havinga molecular weight corresponding to that of NGSP polypeptide. Further,complete clones may be identified by the ability of their inserts, whenplaced in an expression vector, to produce a polypeptide that bindsantibodies specific to the amino-terminal of NGSP polypeptide andantibodies specific to the carboxyl-terminal of NGSP polypeptide.

[0126] Nucleic acid sequences encoding NGSP-derived polypeptides may beproduced by methods well known in the art. In one aspect, sequencesencoding NGSP-derived polypeptides can be derived from NGSP polypeptidecoding sequences by recombinant DNA methods in view of the teachingsdisclosed herein. For example, the coding sequence of NGSP polypeptidemay be altered creating amino acid substitutions that will not affectthe immunogenicity of the NGSP polypeptide or which may improve itsimmunogenicity, such as conservative or semi-conservative substitutionsas described above. Various methods may be used, including but notlimited to oligonucleotide directed, site specific mutagenesis. Theseand other techniques known in the art may be used to create single ormultiple mutations, such as replacements, insertions, deletions, andtranspositions, as described in Botstein and Shortle, 1985, Science229:1193-1210.

[0127] Further, DNA of NGSP polypeptide coding sequences may betruncated by restriction enzyme or exonuclease digestions. Heterologouscoding sequence may be added to NGSP polypeptide coding sequence byligation or PCR amplification. Moreover, DNA encoding the whole or apart of an NGSP-derived polypeptide may be synthesized chemically orusing PCR amplification based on the known or deduced amino acidsequence of NGSP polypeptide and any desired alterations to thatsequence.

[0128] The identified and isolated DNA containing NGSP polypeptide orNGSP-derived polypeptide coding sequence can be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art may be used. Possible vectors include, but are not limitedto, plasmids and modified viruses, but the vector system must becompatible with the host cell used. Such vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas pTrcHis, pBR322 or pUC plasmid derivatives. The insertion into acloning vector can, for example, be accomplished by ligating the DNAfragment into a cloning vector which has complementary cohesive termini.However, if the complementary restriction sites used to fragment the DNAare not present in the cloning vector, the ends of the DNA molecules maybe enzymatically modified. Alternatively, any site desired may beproduced by ligating nucleotide sequences (linkers) onto the DNAtermini; these ligated linkers may comprise specific chemicallysynthesized oligonucleotides encoding restriction endonucleaserecognition sequences. In an alternative method, the cleaved DNA may bemodified by homopolymeric tailing. Recombinant molecules can beintroduced into host cells via transformation, transfection, infection,electroporation, etc., so that many copies of the gene sequence aregenerated.

[0129] In an alternative method, the desired DNA containing NGSPpolypeptide or NGSP-derived polypeptide coding sequence may beidentified and isolated after insertion into a suitable cloning vectorin a “shot gun” approach. Enrichment for the desired sequence, forexample, by size fractionation, can be done before insertion into thecloning vector.

[0130] In specific embodiments, transformnation of host cells withrecombinant DNA molecules that contain NGSP polypeptide or NGSP-derivedpolypeptide coding sequence enables generation of multiple copies ofsuch coding sequence. Thus, the coding sequence may be obtained in largequantities by growing transformants, isolating the recombinant DNAmolecules from the transformants and, when necessary, retrieving theinserted coding sequence from the isolated recombinant DNA.

5.7. Recombinant Production of NGSP Polypeptide and NGSP-DerivedPolypeptides

[0131] NGSP polypeptide and NGSP-derived polypeptides of the inventionmay be produced through genetic engineering techniques. In this case,they are produced by an appropriate host cell that has been transformedby DNA that codes for the polypeptide. The nucleotide sequence encodingNGSP polypeptide or NGSP-derived polypeptides of the invention can beinserted into an appropriate expression vector, i.e., a vector whichcontains the necessary elements for the transcription and translation ofthe inserted polypeptide-coding sequence. The nucleotide sequencesencoding NGSP polypeptide or NGSP-derived polypeptides are inserted intothe vectors in a manner that they will be expressed under appropriateconditions (e.g., in proper orientation and correct reading frame andwith appropriate expression sequences, including an RNA polymerasebinding sequence and a ribosomal binding sequence).

[0132] A variety of host-vector systems may be utilized to express thepolypeptide-coding sequence. These include but are not limited tomammalian cell systems infected with virus (e.g., vaccinia virus,adenovirus, etc.); insect cell systems infected with virus (e.g.,baculovirus); microorganisms such as yeast containing yeast vectors, orbacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA.Preferably, the host cell is a bacterium, and most preferably thebacterium is E. coli, B. subtilis or Salmonella.

[0133] The expression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.In a specific embodiment, a chimeric protein comprising NGSP polypeptideor NGSP-derived polypeptide sequence and a pre and/or pro sequence ofthe host cell is expressed. In other specific embodiments, a chimericprotein comprising NGSP polypeptide or NGSP-derived polypeptide sequenceand an affinity purification peptide is expressed. In further specificembodiments, a chimeric protein comprising NGSP polypeptide orNGSP-derived polypeptide sequence and a useful immunogenic peptide orpolypeptide is expressed. In preferred embodiments, NGSP-derivedpolypeptide expressed contains a sequence forming either anouter-surface epitope or the receptor-binding domain of native NGSPpolypeptide.

[0134] Any method known in the art for inserting DNA fragments into avector may be used to construct expression vectors containing a chimericgene consisting of appropriate transcriptional/translational controlsignals and the polypeptide coding sequences. These methods may includein vitro recombinant DNA and synthetic techniques and in vivorecombinants (genetic recombination). Expression of a nucleic acidsequence encoding NGSP polypeptide or, NGSP-derived polypeptide may beregulated by a second nucleic acid sequence so that the insertedsequence is expressed in a host transformed with the recombinant DNAmolecule. For example, expression of the inserted sequence may becontrolled by any promoter/enhancer element known in the art. Promoterswhich may be used to control expression of inserted sequences include,but are not limited to the SV40 early promoter region (Bernoist andChambon, 1981, Nature 290:304-310), the promoter contained in the 3′long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981,Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences ofthe metallothionein gene (Brinster et al., 1982, Nature 296:39-42) forexpression in animal cells; the promoters of lactamase (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), tac (DeBoeret al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25), _P_(L), or trcfor expression in bacterial cells (see also “Useful proteins fromrecombinant bacteria” in Scientific American, 1980, 242:74-94); thenopaline synthetase promoter region or the cauliflower mosaic virus 35SRNA promoter (Gardner et al., 1981, Nucl. Acids Res. 9:2871), and thepromoter of the photosynthetic enzyme ribulose biphosphate carboxylase(Herrera-Estrella et al., 1984, Nature 310:115-120) for expressionimplant cells; promoter elements from yeast or other fungi such as theGal4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK(phosphoglycerol kinase) promoter, alkaline phosphatase promoter.

[0135] Expression vectors containing NGSP polypeptide or NGSP-derivedpolypeptide coding sequences can be identified by three generalapproaches: (a) nucleic acid hybridization, (b) presence or absence of“marker” gene functions, and (c) expression of inserted sequences. Inthe first approach, the presence of a foreign gene inserted in anexpression vector can be detected by nucleic acid hybridization usingprobes comprising sequences that are homologous to the inserted NGSPpolypeptide or NGSP-derived polypeptide coding sequence. In the secondapproach, the recombinant vector/host system can be identified andselected based upon the presence or absence of certain “marker” genefunctions (e.g., thymidine kinase activity, resistance to antibiotics,transformation phenotype, occlusion body formation in baculovirus, etc.)caused by the insertion of foreign genes in the vector. For example, ifthe NGSP polypeptide or NGSP-derived polypeptide coding sequence isinserted within the marker gene sequence of the vector, recombinantscontaining the insert can be identified by the absence of the markergene function. In the third approach, recombinant expression vectors canbe identified by assaying the foreign gene product expressed by therecombinant. Such assays can be based, for example, on the physical orfunctional properties of NGSP polypeptide or NGSP-derived polypeptide inin vitro assay systems, e.g., binding of a His tag to a column, bindingto an NGSP ligand or receptor, binding with anti-NGSP antibodies of theinvention, or serine protease activity.

[0136] Once a particular recombinant DNA molecule is identified andisolated, several methods known in the art may be used to propagate it.Once a suitable host system and growth conditions are established,recombinant expression vectors can be propagated and prepared inquantity. As explained above, the expression vectors which can be usedinclude, but are not limited to, the following vectors or theirderivatives: human or animal viruses such as vaccinia virus oradenovirus; insect viruses such as baculovirus; yeast vectors;bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNAvectors, to name but a few.

[0137] In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thus,expression of the genetically engineered NGSP polypeptide orNGSP-derived polypeptide may be controlled. Furthermore, different hostcells have characteristic and specific mechanisms for the translationaland post-translational processing and modification of proteins.Appropriate cell lines or host systems can be chosen to ensure thedesired modification and processing of the foreign protein expressed.

5.8. Applications

[0138] The present invention has many utilities. For example, the NGSPpolypeptide and NGSP-derived polypeptides may be used as ligands todetect antibodies elicited in response to Neisseria infections (e.g., asa diagnostic marker in diagnosing Neisseria infections). The NGSPpolypeptide and NGSP-derived polypeptides may also be used as antigensor immunogens for inducing Neisseria-specific antibodies. Suchantibodies are useful in immunoassays to detect Neisseria in biologicalspecimens. The cytotoxic antibodies of the invention are useful inpassive immunizations against Neisseria infections. The NGSPpolypeptide, NGSP-derived polypeptides, and/or fragments thereof mayfurther be used as active ingredients in vaccines to induce an immuneresponse in an animal against Neisseria infections.

[0139] Not intending to be limited to any particular mechanism ofaction, the inventors provide the following remarks. The interaction ofboth normal and neoplastic mammalian cells with extracellular matrixcomponents (ECM) such as fibronectin, vitronectin, and type I collagenhas been shown to be mediated through a family of cell-surface receptorsthat specifically recognize an arginine-glycine-aspartic acid amino acidsequence within each protein (Ruoslahti E. and M. D. Pierschbacher.1986. Arg-Gly-Asp: a versatile cell recognition signal. Cell 44:517-8).Numerous studies have shown that synthetic peptides containing theArg-Gly-Asp sequence can inhibit these receptor-ligand interactions invitro (Gehlsen K. R. Et al. 1988. Inhibition of in vitro tumor cellinvasion by Arg-Gly-Asp-containing synthetic peptides. J. Cell Biol.106:925-30). A highly active Arg-Gly-Asp sequence has been identifiedwithin the cell attachment region of fibronectin and the interactionbetween this sequence and specific platelet cell surface receptors hasbeen demonstrated to induce activation. The conserved Arg-Gly-Asp andArg-Gly-Asn motifs which reside near the C-terminus of the NGSPpolypeptide of the present invention may also function as adherencedomains specific for ECM proteins. If so, once the NGSP polypeptide ofthe present invention is bound to the host's cellular matrix theproteolytic activity of NGSP could function to remodel theepithelial/endothelial surface so as to promote attachment and orsubsequent invasion. Thus using the NGSP polypeptides of the inventionas a vaccine to produce antibody that could interrupt these processeswould be beneficial.

[0140] The polypeptides, peptides, antibodies, nucleic acids and vectorscomprising the nucleic acids, of the invention are useful as reagentsfor clinical or medical diagnosis of Neisseria infections and forscientific research on the properties of pathogenicity, virulence, andinfectivity of Neisseria, as well as host defense mechanisms. Forexample, DNA and RNA of the invention can be used as probes to identifythe presence of Neisseria in biological specimens by hybridization orPCR amplification. The DNA and RNA can also be used to identify otherbacteria that might encode a polypeptide related to the Neisseria NGSP.

[0141] The polypeptides and peptides of the invention may be used toprepare polyclonal and monoclonal antibodies that can be used to furtherpurify compositions containing the polypeptides of the invention byaffinity chromatography. The polypeptides and peptides can also be usedin standard immunoassays as diagnostics to screen for the presence ofantibodies to Neisseria in a sample.

[0142] The nucleic acids, polypeptides and peptides of the invention arealso useful in screening assays to detect compounds, including smallmolecules, or agents that are useful as diagnostic, therapeutic orprophylactic agents against Neisseria infection. In one illustrativemode of this embodiment, assays can be used to screen for a molecule oragent that binds to NGSP and hence which is useful as a diagnostic agentto detect Neisseria in a patient bodily fluid or tissue sample. Inanother illustrative mode of this embodiment, assays can be used toscreen for a molecule or agent that targets NGSP polypeptide or thenucleic acid encoding NGSP polypeptide and hence which molecule or agentis useful as an antibacterial agent for therapy or prophylaxis againstNeisseria infection. While not intending to be limited to any particularmode of action for the antibacterial agents identified according to thepresent invention, the inventors provide the following remarks. Thenovel NGSP polypeptide of the present invention has some limitedsequence similarity to E. coli HtrA or DegP, including, but not limitedto, conserved Arg-Gly-Asp and Arg-Gly-Asn motifs near the C-terminus ofthe NGSP polyp eptide. The inventors envisage that molecules or agentsthat bind to, interact with, or inhibit the synthesis or enzymaticactivity, such as but not limited to, serine protease activity, of theNGSP polypeptide of the invention are useful as anti-infective agentsagainst Neisseria infection. Any assays known to those skilled in theart can be used according to this embodiment to screen for such agents.Non-limiting illustrative examples of assays include the following.

[0143] A number of systems have been described which can be adapted forthe identification of agents interacting with NGSP polypeptide or NGSPderived polypeptides. One well known system is the yeast two-hybridsystem (Fields and Song, 1989, Nature 340:245-246; White. 1996, Proc.Natl. Acad. Sci. USA 93:10001-10003; Warbick, 1997, Structure 5:13-17)which has been used to identify interacting proteins and to isolate thecorresponding encoding genes. In this system, prototrophic selectablemarkers which allow positive growth selection are used as reporter genesto facilitate identification of protein-protein interactions. Applyingthe above general scheme, growing yeast cell colonies expressingDB-X/AD-Y-interacting proteins can be identified among the non-growingcolonies (Gyris et al., 1993, Cell 75:791-803; Durfee et al., 1993,Genes Dev. 7:555-569; Vojtek et al., 1993, Cell 74:205-214). Relatedsystems which may be employed include the yeast three-hybrid system(Licitra and Liu, 1996, Proc. Natl. Acad. Sci. USA 93:12817-12821;Tirode et al., 1997, J. Biol. Chem. 272:22995-22999) and the yeastreverse two-hybrid system (Vidal et al, 1996, Proc. Natl. Acad. Sci. USA93:10321-10326; Vidal et al., 1996, Proc. Natl. Acad. Sci. USA93:10315-10320).

[0144] Bacterial systems for identification of protein-proteininteractions are also known in the art and may be adapted for use withthe methods of the present invention. For example, in one embodiment,the E. coli CadC-based dimer detection system may be used foridentifying proteins interacting with NGSP (see generally, PCTpublication no. WO 99/23116 dated May 14, 1999, which is incorporatedherein in its entirety). In another embodiment, a bacterial proteininteraction system based on the AraC protein, which regulates theL-arabinose operon in E. coli, may be used (Bustos and Schleif, 1993,Proc. Natl. Acad. Sci. USA 90:5638-5642; Soisson et al., 1997, Science276:421-425; Eustance et al., 1994, J. Mol. Biol. 242:330-338). Otherassay systems which may be used include bacterial systems based on thelambda repressor system (Zeng et al., 1997, Protein Sci. 6:2218-2226),the lac-operon (Gates et al., 1996, J. Mol. Biol. 255:373-386), aninteraction signal detection based on lambda and lambdoid proteins(Hollis et al., 1988. Proc. Natl. Acad. Sci. USA 85:5834-5838), systemsbased on E. coli RNAP (Dove et al., 1998, Genes Dev. 12:745-754; Dove etal., 1997, Nature 386:627-630), and systems based on the cAMP synthetase(Karimova et al., 1998, Proc. Natl. Acad. Sci. USA 95:5752-5756).

[0145] Alternatively, assays screening for interaction of molecules withNGSP can be devised using a detectible marker. Proteins or othermolecules may be labeled with a detectable marker using methods forprotein labeling known in the art. A “detectable marker” refers to amoiety, such as a radioactive isotope or group containing same, ornonisotopic labels, such as enzymes, biotin, avidin, streptavidin,digoxygenin, luminescent agents, dyes, haptens, and the like.Luminescent agents, depending upon the source of exciting energy, can beclassified as radioluminescent, chemiluminescent, bioluminescent, andphotoluminescent (including fluorescent and phosphorescent). An affinitycapture assay may be used.

[0146] In another embodiment, any molecule including macromolecules andsmall molecules, can be assayed for interaction with NGSP polypeptide oran NGSP-derived polypeptide; interaction with NGSP or an NGSP-derivedpolypeptide indicates the molecule is useful as a diagnostic,therapeutic or prophylactic against Neisseria infection. In oneembodiment, the method is as follows. A method for assaying for an agentthat interacts with NGSP polypeptide comprises: (a) contacting a cellexpressing NGSP polypeptide with an agent labeled with a detectablemarker for a time sufficient to allow the agent to interact with thepolypeptide; (b) washing the cells; and (c) detecting any markerassociated with the cells, in which any cell associated marker indicatesthat the agent interacts with the NGSP polypeptide and wherein any agentthat interacts with NGSP indicates that the agent is useful as adiagnostic, prophylactic or therapeutic agent against Neisseriainfection.

[0147] DNA or polypeptides of the invention may be used to assess thebinding of small molecule substrates and ligands in, for example, cells,cell free preparations, chemical libraries, and natural product extractsand mixtures. These substrates and ligands may be natural substrates andligands or may be structural or functional mimetics thereof.

[0148] The invention also provides a method of screening compounds toidentify those which enhance (i.e., agonists) or block (i.e.,antagonists) of the action of NGSP polypeptides, particularly thosecompounds that are bacteriostatic or bactericidal. The method ofscreening may involve high-throughput assay techniques. For example, toscreen for agonists or antagonists, a synthetic reaction mix, a cellularcompartment, such as a membrane, cell envelope or cell wall, or apreparation of any mixture thereof, comprising NGSP polypeptide and alabeled substrate or ligand of such polypeptide is incubated in theabsence or the presence of a candidate molecule that may be a NGSPagonist or antagonist. The ability of the candidate molecule to agonizeor antagonize the NGSP polypeptide is reflected in decreased binding orthe labeled ligand or decreased production of product from suchsubstrate. Molecules that bind gratuitously, ie., without inducing theeffects of NGSP polypeptide are most likely to be good antagonists.Molecules that bind well and increase the rate of product productionfrom substrate are agonists. Detection of the rate or level ofproduction of product from substrate may be enhanced by using a reportersystem. Reporter systems that may be useful in this regard include butare not limited to colorimetric labeled substrate converted intoproduct, a reporter gene that is responsive to change in a NGSPpolypeptide activity, and binding assays known in the art. Potentialantagonists or agonists include small molecules, peptides, andantibodies that bind to a NGSP peptide or polypeptide or the invention,or such a closely related protein or antibody that binds the same siteson a binding molecule.

[0149] It is to be understood that the application of the teachings ofthe present invention to a specific problem or environment will bewithin the capabilities of one having ordinary skill in the art in lightof the teachings contained herein.

[0150] The above disclosure generally describes the present invention. Amore specific description of certain embodiments is provided below inthe following examples. The examples are described solely for thepurpose of illustration and are not intended to limit the scope of theinvention. Changes in form and substitution of equivalents arecontemplated as circumstances suggest or render expedient. Althoughspecific terms have been employed herein, such terms are intended in adescriptive sense and not for purposes of limitation.

[0151] Methods of molecular genetics, protein biochemistry andimmunology used but not explicitly described in the disclosure andexamples are amply reported in the scientific literature and are wellwithin the ability of those skilled in the art.

6. EXAMPLE Isolation and Characterization of the NGSP Polypeptide andSequence Encoding Same 6.1. Extraction of Envelope Proteins

[0152] Neisseria are grown at 37° C. at 200 rpm in 1 liter of MuellerHinton broth, chocolate agar plates or Columbia blood agar plates.Extraction with hypotonic solutions is carried out as follows. Cells areharvested into lithium chloride (LiCl), sodium acetate (NaOAc) solution(0.1M LiCl, 0.2 M NaOAc, pH 5.8) and shaken with glass beads for 3 h ina 45° C. water bath. The beads and cellular debris are removed bycentrifugation and for crude extracts, the supernatant removed andstored at −20° C. For purified extracts, the supernatant is furthercentrifuged at 100,000×g and the resulting pellet resuspended and eitherstored or used for further purification as described herein.

[0153] Extraction using detergents is carried out as follows. Cells areharvested into a Tris-hydrochloride buffer solution and pelleted bycentrifugation. The pelleted cells are resuspended and sonicated todisrupt the cells. Unbroken cells are removed by low speedcentrifugation and the total cell envelope fraction is treated witheither (1.25 % final w/v) n-octyl-D- glucopyranoside (ie., octylglucoside; OG) in phosphate buffered saline (PBS) or (0.5 % w/v) ofsodium N-lauroyl sarcosine (Sarkosyl) for 30 minutes at roomtemperature. The unsolublized fraction is pelleted and the supernatantis used as the detergent extract for resolution using SDS-PAGE or forfurther purification as described herein.

6.2. Amino Terminal Sequencing of NGSP Polypeptide

[0154] NGSP polypeptide from extracts of Neisseria is detected (e.g., bysilver staining or anti-NGSP antibodies) in denaturing gels. ForN-terminal sequencing, an extract is mixed with PAGE sample buffercontaining SDS, and is incubated for 3 minutes in boiling water bath.The proteins are then resolved on a PAGE with SDS and transferred to aPVDF membrane by electroblotting. The region of the membrane containingthe NGSP band is then cut out and amino-terminal sequencing is performedby generally accepted methods known to those skilled in the art.

6.3. Anti-NGSP Antiserum

[0155] Antisera to NGSP are prepared by injecting the NGSP polypeptideinto an animal, such as a rabbit, mouse or guinea pig, with or withoutan adjuvant. For instance, NGSP is injected with Freund's completeadjuvant followed by injections of NGSP with Freund's incompleteadjuvant. Normally, an isolated, a semi-purified or purified form of theprotein is injected. For instance, the NGSP polypeptide is resolved fromother proteins using a denaturing sodium dodecylsulfqte polyacrylamidegel according to standard techniques well known to those skilled in theart, as previously described (Laemmli, 1970, Nature 227:680-685), andcutting the NGSP-containing band out of the gel. The excised bandcontaining NGSP is macerated and injected into an animal to generateantiserum to the NGSP polypeptide. The antisera is examined using wellknown and generally accepted methods of ELISA to determine titres, bywestern blots to determine binding to proteins, for immunofluorescentstaining and for cpmplement-mediated cytotoxic activity againstNeisseria as described below.

6.4. Western Blots

[0156]Neisseria gonorrhoeae (clinical isolate) is grown on gonococcalagar (GC agar base, Difco; supplemented with 1%. Iso Vitale X, BBL) orchocolate agar plates for 24-48 hours at 37° C. in 5% CO₂. Cells areremoved by scraping the colonies from the agar surface using apolystyrene inoculating loop. Cells are then solubilized by suspending30 μg of cells in 150 μl of PAGE sample buffer (360 mM Tris buffer [pH8.8], containing 2-mercaptoethanol, 4% sodium dodecylsulfate and 20%glycerol), and incubating the suspension at 100° C. for 5 minutes. Thesolubilized cells are resolved on 12% polyacrylamide gels as per Laemmliand the separated proteins were electrophoretically transferred to PVDFmembranes at 100 V for 1.5 hours as previously described (Thebaine etal. 1979, Proc. Natl. Acad. Sci. USA 76:4350-4354). The PVDF membranesare then pretreated with 25 ml of Dulbecco's phosphate buffered salinecontaining 0.5% sodium casein, 0.5% bovine serum albumin and 1% goatserum. All subsequent incubations are carried out using thispretreatment buffer.

[0157] PVDF membranes are incubated with 25 ml of a 1:500 dilution ofpreimmune rabbit serum or serum from a rabbit immunized with NGSP orHin47 polypeptide (as described above) for 1 hour at room temperature ormonoclonal antibodies to NGSP or to Hin47 (described above). PVDFmembranes are then washed twice with wash buffer (20 mM Tris buffer [pH7.5.] containing 150 mM sodium chloride and 0.05% Tween-20). PVDFmembranes are incubated with 25 ml of a 1:5000 dilution ofperoxidase-labeled goat anti-rabbit (or anti-mouse for monoclonals) IgG(Jackson ImmunoResearch Laboratories, West Grove, Pa.) for 30 minutes atroom temperature. PVDF membranes are then washed 4 times with washbuffer, and are developed with 3,3′ diaminobenzidine tetrahydrochlorideand urea peroxide as supplied by Sigma Chemical Co. (St. Louis, Mo.catalog number D-4418) for 4 minutes each.

6.5. Anti-NGSP Immunofluorescence Staining of Cell Surface

[0158] Neisseria are grown overnight at 37° C. in a shaking water bathin Mueller Hinton broth or on gonoccal agar and harvested by scraping.The cells are pelleted by centrifugation and then resuspended in anequal volume of Dulbecco's modification of phosphate buffered salinewithout calcium or magnesium (PBS/MC). 20 μl of the cell suspension isapplied to each of 5 clean microscope slides. Afier setting for 10seconds, the excess fluid is removed with a micropipettor, and theslides are allowed to air dry for 1 hour. The slides are then heat fixedover an open flame until the glass is warm to the touch. The slides areinitially treated with 40 μl of 1:40 dilution of anti-NGSP antiserum orpreimmune serum from the same animal diluted in PBS/MC, or PBS/MC for 10minutes, then washed 5 times with PBS/MC. The slides are treated with 40μl of 5 μg/ml PBS/MC of fluorescein isothiocyanate-labeled goat antibodyto rabbit IgG (Kirkegaard and Perry Laboratories, Inc, Gaithersburg,Md). The slides are incubated in the dark for 10 minutes and are washed5 times in PBS/MC. Each slide is stored covered with PBS/MC under acover slide and is viewed with a fluorescence microscope fitted with a489 nm filter. For each sample five fields-of-view are visually examinedto evaluate the extent of straining.

6.6. Cellular Envelope Location of NGSP

[0159] Rabbit anti-NGSP antiserum is used in indirect immunofluorescencestaining to determine if NGSP polypeptide is exposed on the outersurface of Neisseria cells. This would indicate that in intact Neisseriacells NGSP polypeptide is reactive with anti-NGSP antibodies.

6.7. Properties of NGSP Polypeptide

[0160] NGSP polypeptide exists as a protein of approximately 40-55 kD inits native state as can be determined using detergent or hypotonicextracts of Neisseria, incubating the extracts with sodium dodecylsulfate at 100° C., and resolving the proteins on a denaturingpolyacrylamide gel.

[0161] Western blot analysis of protein extracts of a number ofNeisseria strains can be used to show that the anti-NGSP antibodies bindto a polypeptide of about 40 kD to about 55 kD in many Neisseriastrains. Anti-NGSP antibodies may be used to specifically identifyNeisseria. NGSP polypeptide may be used to generate antibodies that havediagnostic application for identification of Neisseria. Antibodies toNGSP polypeptide of one species or strain may be used to identify andisolate the corresponding NGSP polypeptide of other Neisseria species orstrains.

7. EXAMPLE Efficacy of NGSP Vaccine Cytotoxic Activity of Anti-NGSPAntiserum

[0162] Complement-mediated cytotoxic activity of anti-NGSP antibodies isexamined to determine the vaccine potential of NGSP polypeptide.Antiserum to NGSP polypeptide is prepared as described in Section 6.3.supra. The activities of the pre-immune serum and the anti-NGSPantiserum in rmediating complement killing of Neisseria are examinedusing a “Serum Bactericidal Test,” for instance, as described byZollinger et al. (Immune Responses to Neisseria meningitis, in Manual ofClinical Laboratory Immunology, 3rd ed., pg 347-349). The results areused to show that anti-NGSP antiserum mediates complement-killing ofNeisseria.

8. EXAMPLE Isolation of the NGSP Nucleic Acid Sequence 8.1.Identification of a NGSP Open Reading Frame

[0163] The E.coli DegP (HtrA) amino acid sequence available fromGeneBank was employed as a BLAST (TBLASTN) subject query to search thegenomic sequence databases for N. gonorrhoeae (Univ. Oklahoma, USA)strain 1090 to identify linear amino acid sequences that might sharesome similarity to the DegP protein. No predicted amino acid sequencesfrom this Neisseria databases showed more than ˜30-35% similarity to theE. coli DegP protein sequence. Candidate N. gonorrhoeae NGSP amino acidsequences were localized within specific genomic DNA sequence “contigs”,and putative open reading frames encoding these NGSP sequences werederived. Putative ORFs capable of encoding proteins of ˜40-55 kD, theaverage size of most DegP-like serine proteases, were then selected andfurther analyzed for the presence and appropriate relative spacing ofsemi-conserved catalytic residues (H, D, S) thought to be required forserine protease activity. A single putative open reading frame from theN. gonorrhoeae database was identified which met these criteria.

8.2. Isolation of N. Gonorrhoeae Chromosomal DNA

[0164]N. gonorrhoeae strain GC340 was obtained from the Centers forDisease Control and Prevention (CDC). GC340 was streaked on gonococcalagar base (GC agar, Difco) containing 1.0% Iso Vitale X (BBL) and grownat 35-37° C. in 5% CO₂ for ˜24-28 hours. To prepare confluent “lawns” ofcells for DNA isolation, three or four single colonies were picked fromthe “overnight” seed plate and used to inoculate fresh GC plates whichwere again grown overnight at 35-37° C. in 5% CO₂. Cells were collectedfrom the surface of the agar plates by gentle rinsing using trypticasesoy broth (TSB) containing 10% glycerol and then stored at −20° C. Whenneeded, cells, were thawed at room temperature and bacteria collected bycentrifugation in a Sorval SS34 rotor at ˜2000×g for 15 minutes at roomtemperature. The supernatant was removed and the cell pellet suspendedin ˜5.0 ml of sterile water. An equal volume of lysis buffer (200 mMNaCl, 20 mM EDTA, 40 mM Tris-HCl pH8.0, 0.5% (w/v) SDS, 0.5% (v/v)2-mercaptoethanol, and 250 ug/ml of proteinase K) was added and thecells suspended by gentle agitation and trituration. The cell suspensionwas then incubated ˜12 hours at 50° C. to lyse the bacteria and liberatechromosomal DNA. Proteinaceous material was precipitated by the additionof 5.0 ml of saturated NaCl (˜6.0M, in sterile water) and centrifugationat ˜5,500×g in a Sorval SS34 rotor at room temperature. Chromosomal DNAwas precipitated from the cleared supernatant by the addition of twovolumes of 100% ethanol. Aggregated DNA was collected and washed usinggentle agitation in a small volume of a 70% ethanol solution. Purifiedchromosomal DNA was suspended in sterile water and allowed todissolve/disburse overnight at 4° C. by gentle rocking. Theconcentration of dissolved DNA was determined spectrophotometrically at260 nm using an extinction coefficient of 1.0 O.D. unit ˜50 ug/ml.

8.3. PCR Cloning of the NGSP ORF

[0165] To produce high levels of recombinant N. gonorrhoeae NGSP proteinfor immunogenicity and protective efficacy studies the NGSP ORF was PCRcloned into an E.coli high expression vector under the control of thetrc promoter. Oligonucleotide PCR primers complementary to the DNAsequences encoding the first 10 amino acid residues of the N-terminus(VFKKYQYFAL) (SEQ ID NO: 9) and the last 11 C-terminal amino acidresidues (EILAVRASPRQ) (SEQ ID NO: 10) of the N. gonorrhoeae NGSP ORFpresent in the Oklahoma genomic database were synthesized. In additionto the NGSP specific sequences, these PCR primers were designed tocontain flanking EcoRI and SalI restriction sites, respectively, in aneffort to expedite directional cloning of the ORF into the expressionvector pTLZ2. These oligonucleotides were used to amplify NGSP-specificPCR products from the clinically relevant N.gonorrhoeae strain GC340.

[0166] The amplification primers used for these PCR reactions weredesignated NgH47-Fn/t-RI (46 mer, forward primer) and NgH47-RCh/t-Sal(69 mer, reverse primer). In addition to the NGSP coding sequences, theforward primer was designed to contain a unique EcoRi restriction sitelocated upstream of the NGSP ORFs Met initiation residue. The reverseprimer was designed to contain six contiguous His codons (CAT or CAC)immediately downstream and in the same translational reading frame asthe 3′ NGSP coding sequence. A unique SalI restriction site wasengineered into the reverse primer downstream and adjacent to atranslatoin stop codon (TAA) that follows the last His codon tofacilitate directional cloning into pTLZ2. Thus recombinant NGSP proteinexpressed from the insert amplified using the NgH47-Fn/t-RI andNgH47-RCh/t-Sal primers will carry a (His)₆ affinity purification tag atthe C-terminus. NgH47-Fn/t-RI (46 mer) 5′-AGG CAG AGG GAA TTC ATG TTCAAA (SEQ ID No. 1) AAA TAC CAA TAC TTC GCT TTG G-3′ NgH47-Rch/t-Sal (69mer) 5′-AGG CAG AGG GTC GAC TAA ATG GTG ATG GTG ATG GTG TTG ACG GGG ACTTGC (SEQ ID No. 2) CCT GAC GGC TAG GAT TTC-3′

[0167] Standard PCR amplification reactions (2 mM Mg²⁺, 200 umol dNTPs,0.75 nits AmpliTaq, 50 ul final volume) were programmed using ˜0.1 ug ofN. gonorrhoeae GC340 chromosomal DNA. Amplification of the NGSP targetsequence was achieved using a standard 32-cycle, three-step thermalprofile, i.e. 95° C., 30 sec; 60° C., 45 sec, 72° C., 1 min.Amplification was carried out in 0.2 ml polypropylene thin-walled PCRtubes (Perkin-Elmer) in a Perkin-Elmer model 2400 thermal cycler. PCRamplification reactions produced the expected NGSP-specific ˜1.4 Kbpamplimer.

[0168] The ˜1.4 Kbp NGSP amplimer was purified from unincorporatedprimers using hydroxyapatite spin columns (QiaGen) and digested tocompletion with an excess of EcoRI and SalI (BRL, ˜10 units per 1 ugDNA) according to the manufacturers recommendations. The purified anddigested NGSP ORF was then purified via QiaGen columns as describedabove and cloned into plasmid pTLZ2 that had been previously digested tocompletion with both EcoRI and SalI and treated with calf intestinalalkaline phosphatase (BRL, ˜0.05 units/pmole of 5′ ends) to preventvector religation (˜5:1, insert:vector ratio).

[0169] Digestion of the ˜7.9 Kbp pTLZ2 vector with EcoRI and SalIproduces two restriction fragments; one a ˜3.1 Kbp fragment encoding theE.coli β-galactosidase gene and the other a ˜4.8 Kbp plasmid repliconcontaining the pBR322 origin of replication, an ampicillin resistancegene, the E.coli lacI^(q) repressor for controlled expression ofexogenous cloned genes, the procaryotic rrnB transcriptional stopsequence, and the highly efficient and regulated tac promoter.

[0170] Aliquots from the ligation reaction were then used toelectrotransform a suitable E.coli host (e.g. JM109). Transformed cellswere plated on 2X-YT agar containing 100 ug/ml ampicillin and grown at37° C. for ˜12-16 hours. Mini-prep DNA from ampicillin-resistanttransformants picked at random were prepared using commerciallyavailable reagents (QiaGen Mini Prep Kit) and examined for the presenceof recombinant plasmids larger than the ˜4.8 Kbp vector plasmid pTLZ2(i.e. insert-carrying plasmids). Putative NGSP-insert carryingrecombinant plasmids were then digested to completion with EcoRI andSalI and examined for the presence of the ˜1.4 Kbp NGSP-specificfragment by standard agarose gel electrophoresis (0.8% agarose, TAEbuffer). All ˜6.2 Kbp plasmids tested were found to contain the NGSPinsert. Plasmid pTLZ-NgHtrA #2 was one recombinant derivative isolatedby these procedures. A map of pTLZ-Ng HtrA#2 NGSP is presented in FIG.1.

8.4. Expression of Recombinant NGSP Protein

[0171] The ability of pTLZ-NgHtrA #2 to express the N. gonorrhoeaerecombinant NGSP protein was assessed by SDS-PAGE. A 5.0 ml overnightculture of JM109 (pTLZ-NgHtrA #2) was prepared in LB broth containingampicillin (100 ug/ml) and inoculated with cells from a “patch” platemade directly from the original pTLZ-NgHtrA #2 transformant colony andgrown overnight at 37° C. with shaking (˜250 rpm). An aliquot of theovernight seed culture (˜1.0 ml) was inoculated into a 125 ml erlenmeyerflask containing ˜25 of LB/Ap broth and grown at 37° C. with shaking(˜250 rpm) until the culture turbidity reached O.D.600 of ˜0.5, i.e.mid-log phase (usually about 1.5-2.0 hours). At this time approximatelyhalf of the culture (˜12.5 ml) was transferred to a second 125 ml flaskand expression of recombinant NGSP protein induced by the addition ofIPTG (1.0 M stock prepared in sterile water, Sigma) to a finalconcentration of 1.0 mM. Incubation of both the IPTG-induced andnon-induced cultures continued for an additional ˜4 hours at 37° C. withshaking.

[0172] Samples (˜1.0 ml) of boqth induced and non-induced cultures wereremoved after the induction period and the cells collected bycentrifugation in a microcentrifuge at room temperature for ˜3-5minutes. Individual cell pellets were suspended in ˜50 μl of sterilewater, then mixed with an equal volume of 2× Laemmli SDS-PAGE samplebuffer containing 2-mercaptoethanol, and placed in boiling water bathfor ˜3-5 min to denature and reduce the recombinant protein. Equalvolumes (˜15 μl) of both the crude IPTG-induced and the non-induced celllysates were loaded onto duplicate 4-20% Tris/glycine polyacrylamidegradient gels (1 mm thick Mini-gels, Novex).

[0173] The induced and non-induced lysate samples were electrophoresedtogether with prestained molecular weight markers (SeeBlue, Novex) underconventional electrophoresis conditions (˜30 mA, constant current) usinga standard SDS/Tris/glycine running buffer (BioRad). Followingelectrophoresis, one gel was stained with commassie brilliant blue R250(BioRad) and then destained using an acetic acid:methanol:waterdestaininrg solution to visualize novel ˜50 kDa NGSP arabinose-inducibleprotein.

[0174] The second gel was electroblotted onto a PVDF membrane (0.45micron pore size, Novex) for ˜2 hr at 4° C. using a BioRad Mini-ProteanII blotting apparatus and Towbin's methanol (20%) transfer buffer.Blocking of the membrane and antibody incubations were performed using aTris (50 mM,pH7.3):CaCl₂ (1 mM):Tween-20 (0.2%) buffer containing 0.5%casein. A monoclonal anti-(His)₅ antibody conjugated to HRP (QiaGen) wasused at a 1/5,000 dilution to confirm the expression and identify of ˜50kDa inducible rNGSP protein. Visualization of the antibody reactivepattern was achieved on Hyperfilm using the Amersham ECLchemiluminescence system. The results from the Western blot experimentare shown in FIG. 2.

8.5. Purification of Recombinant Protein

[0175] Recombinant NGSP protein is purified to homogeneity usingstandard preparative column chromatographic procedures. Briefly, an E.coli strain harboring the expression plasmid pTLZ-NgHtrA #2 is grown inLuria broth in a 5 l fermenter (New Brunswick) at 37° C. with moderateaeration until mid-log phase (˜0.5 O.D.₆₀₀) and induced with IPTG (1 mmfinal) for 4-5 hours. Cell paste is collected, washed in PBS and storedat −20° C. Aliquots of frozen cell paste (˜9-10 g wet weight) aresuspended in ˜120 ml of D-PBS by mechanical agitation and lysed bypassage through a French pressure cell (2×, 14,000 psi, 4° C.). Theexact sample preparation methodology to be used for NGSP purificationvaries somewhat depending on whether the NGSP protein is expressed as asoluble component or as insoluble inclusion bodies.

[0176] A general process for the purification of NGSP protein as asoluble protein is given below. Insoluble material is removed afterFrench press disruption by high speed centrifugation (˜10,000×g, 4° C.,30 min). The soluble fraction containing NGSP is suspended in ˜20 ml ofice cold 50 mM Tris-HCl buffer (pH8.0) and loaded onto a DEAE-Sephacel(Pharmacia) ionic exchange column (˜5 cm×60 cm). To minimizeautoproteolysis of the NGSP protein, chromatography is conducted at 4°C. Unbound material is washed from the column using loading buffer (50mM Tris-Hcl, pH8.0) prior to elution of bound NGSP protein. Elution ofNGSP from the Sephacel matrix is achieved using a NaCl gradient(0.05-0.5M NaCl, in 50 mM Tris-Hcl, pH8.0). Fractions released by thesalt gradient are collected and examined by standard SDS-gelelectrophoresis methodologies for the presence of a ˜40-55 kd protein.Fractions are also assayed for protease activity using a standardazocasein colorimetric assay. Fractions containing NGSP are pooled andextensively dialyzed against 10 mM sodium phosphate buffer (SPB, pH8.0)at 4° C.

[0177] The partially purified NGSP is then applied to a hydroxylapatitecolumn, previously equilibrated in SPB. Bound proteins are eluted usinga 0.1-0.5M NaCl gradient in SPB. Fractions are collected periodicallyduring elution and examined for the presence of NGSP by SDS-gelelectrophoresis and protease activity as above. Eluted material isdialyzed against 50 mM Tris-HCl to remove residual salt and concentratedusing a Centricon-30 concentrator (Amicon, 30,000 MWCO).

8.6. Generation of a Radiolabelled Screening Probe

[0178] The sequence information shown above is used to design a pair ofnondegenerate convergent (i.e., one forward and one reverse primer)oligonucleotide primers. PCR amplification of DNA fragments is performedunder the same conditions as described above with the exception that theannealing temperature is raised to 50° C. The DNA fragment is isolatedfrom an agarose gel as before and radiolabelled using [32P]-gamma-ATPand T4 polynucleotide kinase according to standard methods.Unincorporated radiolabel is separated from the probe on a G25 Sepharosespin colurmn. Before use, the probe is denatured for 2 min. at 95° C.and subsequently chilled on ice (4° C).

8.7. Hybridization of Plaque-Lift Filters and Southern Blots withRadiolabelled Probe

[0179] Phage plaques from library platings are immobilized on nylonfilters using standard transfer protocols well known to those skilled inthe art. Digested bacterial genomic DNA, phage or plasmid DNA iselectrophoresed on 0.8% TAE-agarose gels and transferred onto nylonfilters using a pressure blotter (Stratagene) according to themanufacturer's recommendations. Hybridizations with selected probes areperformed at 37° C. Hybridizations with other probes are generallycarried out at 60° C. Washes of increasing stringency are done at therespective hybridization temperatures until nonspecific background isminimized.

8.8. Construction of a Neisseria Gonorrhoeae Genomic DNA Library

[0180] A genomic library is constructed in the λZAPII replacement vectorobtained from Stratgene. The vector arms are digested with EcoR1.Digests of Neisseria gonorrhoeae DNA by EcoR1 are performed to yieldfragment sizes between 1 kb and 5 kb. Ligations of vector arms andinsert DNA are carried out according to standard protocols. Ligationreactions are packaged in vitro using the Stratagene GigaPack Gold IIIextract. The packaged phage are plated on E. coli X1 Blue MRA (P2)(Stratagene). An initial library titer is determined and expressed asnumber of pfu.

[0181] The library is screened using 4×10⁴ pfu that are plated at adensity of 8×10³ pfu/130 mm plate. Several putative positive phageplaques are identified by screening the library with a radiolabelledNGSP-specific DNA hybridization probe or a NGSP-monospecific antibodyand the strongest hybridizing phage are eluted from cored agarose plugs,titered and replated for secondary screening. The selected phages arereplated at low density (approximately 100 pfu/plate) and plaques areanalyzed by PCR using primer pairs. Inserts carrying plasmids(phagemids) are rescued from the selected phage by co-infecting E. colicells with an appropriate helper virus.

8.9. Determination of Insert Size and Mapping of DNA Fragments

[0182] In order to estimate the size of inserts, phagemid DNA isdigested with NotI and the digests are analyzed on a 0.5% TAE-agarosegel side by side with suitable DNA markers. In order to map restrictionfragments that would hybridize to the probe, DNA from phagemid isolatesis digested with a number of common restriction enzymes either alone orin combination with NotI. The rationale of this approach is todiscriminate between fragments that span the insert/phagemid vectorjunction and those that map on the NotI insert. The series of single anddouble digests are run side-by-side for each phage isolate and analyzedby Southern analysis with radiolabelled probe.

9. EXAMPLE Sequencing of the NGSP Nucleic Acid

[0183] Sequencing of the NGSP encoding nucleic acid from pTLZ-NgHtrA#2is performed using the plasmid pTLZ-NgHtrA#2 as a template. Allsequencing reactions are performed using the Dye Terminator CycleSequencing Kit from Perkin-Elmer according to the manufacturer'sspecifications. The sequencing reactions are read using an ABI Prism 310Genetic Analyzer. The sequences are aligned using the AutoAssemblersoftware (Perkin-Elmer) provided with the ABI Prism 310 sequencer. Thisplasmid was inserted into E. coli JM109 (Invitrogen) and deposited withAmerican Type Culture Collection (ATCC) as E. coli JM109(pTLZ-NgHtrA#2).

[0184] The nucleotide sequence of the NGSP gene is shown in SEQ ID NO:3.A deduced amino acid sequence of the open reading frame of NGSP is shownin SEQ ID NO:4.

10. EXAMPLE Genetic Analysis 10.1. Knock-Out Mutants

[0185] A genomic knock-out mutation of the NGSP gene is constructedusing standard methodologies. For example, the NGSP gene from strainGC340 which has been cloned into a suitable plasmid vector, e.g.,plasmid pTLZ-NgHtrA#2, is digested with a restriction enzyme (e.g.,AscI) that cuts the NGSP gene only once. The digested NGSP plasmid isthen ligated to a DNA fragment encoding a suitable resistance marker,e.g., the kanamycin resistance (KAN^(R)) cassette from plasmid pUC4-K.The ligation mixture is then used to transform E. coli cells to KAN^(R).Once the presence of the KAN^(R)-insert is confirmed by restrictionanalysis, these cloned NGSP KAN^(R)-derivatives are used to transformcompetent N. gonorrheoeae. Although N. gonorrhoeae are naturallycompetent, standard procedures are used to enhance transformationefficiency. Transformants are analyzed by Southern blotting and/or PCRto identify knockout mutants that have recombined the NGSP-KAN^(R)cassette into the chromosome.

10.2. PCR Analysis

[0186] DNA from KAN^(R) Neisseria gonorrhoeae colonies is analyzed byPCR using primers that hybridize to flanking sequences upstream anddownstream of the NGSP gene. A PCR product equal in size to the nativegene is only to be expected if the incoming targeting cassette has notbeen integrated into the genome by homologous recombination.Amplification products longer than the native gene are obtained onlywhen the KanR cassette has been successfully integrated.

10.3. Southern Analysis of NGSP

[0187] Genomic DNA from wild-type Neisseria gonorrhoeae and from PCRpositive deletion mutants is digested with EcoRI. The digests areseparated on a 0.8% TAE-agarose gel and transferred to nylon membranesusing standard protocols. The blots are hybridized with ³²P labeledprobes prepared from either the NGSP region or from the Kanarmycinresistance gene of pTLZ-NgHtrA#2. Using the NGSP probe, fragments havingappropriate sizes are detected in the EcoRI digests on DNA from allwild-type strains tested, whereas DNA fragments roughly 1.2 kbp longerare detected in digests on DNA from the knockout mutants. The presenceof this unique, new restriction fragment demonstrates the successfultargeting of the NGSP locus.

[0188] Probing of the membrane with the kanamycin gene does not generateany signal in Neisseria gonorrhoeae wild-type DNA. In DNA from theknockout mutants, the kanamycin probe detects fragments havingappropriate sizes in EcoRI digests. The presence of these sequences inthe deletion mutants and their absence in the wild-type DNA demonstratesthat the NGSP locus is successfully altered.

11. EXAMPLE Generation and Reactivity of Monoclonal Anti-NGSP Antibodies

[0189] BALB/c mice are immunized with total outer membranes fromNeisseria or with NGSP. Hybridomas for monoclonal antibodies areprepared by fusing the spleen cells from these mice to SP2/0 cells andselecting for successful hybrids with HAT containing media. Reactivehybridomas are screened using an ELISA containing detergent extracts ofthe total outer member of Neisseria. From this screen, hybridomas withvarying levels of activity in the ELISA are selected for clonalselection, the monoclonal antibodies are assayed for reactivity topurified NGSP and total outer membranes from Neisseria by ELISA.Monoclonal antibodies are selected that react specifically to NGSP inthe ELISA.

[0190] Western blots are performed as described in Example 6.4., usingmonoclonal antibodies.

12. DEPOSIT OF MICROORGANISM

[0191]E. coli JM 109 containing plasmid pTLZ-NgHtrA#2 was deposited onAug. 5, 1999 with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas Va., 20110-2209, USA, under theprovisions of the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedures, andassigned number PTA-470.

[0192] The present invention is not to be limited in scope by themicroorganism deposited or the specific embodiments described herein. Itwill be understood that variations which are functionally equivalent arewithin the scope of this invention. Indeed, various modifications of theinvention, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the foregoing description andaccompanying drawings. Such modifications are intended to fall withinthe scope of the appended claims.

[0193] Various publications are cited herein, the disclosures of whichare incorporated by reference in their entireties.

1 10 1 46 DNA Artificial Sequence Description of Artificial SequencePrimer 1 aggcagaggg aattcatgtt caaaaaatac caatacttcg ctttgg 46 2 69 DNAArtificial Sequence Description of Artificial Sequence Primer 2aggcagaggg tcgacttaat ggtgatggtg atggtgttga cggggacttg ccctgacggc 60taggatttc 69 3 1395 DNA Neisseria gonorrhoeae 3 gtgttcaaaa aataccaatacttcgctttg gcggcactgt gtgccgcctt gctggcaggc 60 tgcgaaaagg caggcagctttttcggtgcg gacaaaaaag aagcatcctt cgtagaacgc 120 atcgaacaca ccaaagacgacggcagtgtc agtatgctgc tgcccgactt tgcccaactg 180 gttcaaagcg aaggcccggcagtcgtcaat attcaggcag cccccgcccc gcgcacccaa 240 aacggcagcg gcaatgccgaaaccgattcc gacccgcttg ccgacagcga cccgttctac 300 gaatttttca aacgcctcgtcccgaacatg cccgaaatcc cccaagaaga agcagatgac 360 ggcggattga acttcggttcgggcttcatc atcagcaaaa acggctacat cctgaccaat 420 acccacgtcg ttgccggtatgggcagtatc aaagtcctgc tcaacgacaa gcgcgaatat 480 accgccaaac tcatcggttcggatgtccaa tccgatgtcg cccttctgaa aatcgacgca 540 acggaagagc tacccgtcgtcaaaatcggc aatcccaaaa atttgaaacc gggcgaatgg 600 gtcgctgcca tcggcgcgcccttcggcttt gacaacagcg tgaccgccgg catcgtgtcc 660 gccaaaggca gaagcctgcccaacgaaagc tacacaccct tcatccaaac cgacgttgcc 720 atcaatccgg gcaattccggcggcccgctg ttcaacttaa aaggacaggt cgtcggcatc 780 aattcgcaaa tatacagccgcagcggcgga ttcatgggca tctcctttgc catcccgatt 840 gacgttgcca tgaatgtcgccgaacagctg aaaaacaccg gcaaagtcca acgcggacaa 900 ctgggcgtga ttattcaggaagtatcctac ggtttggcac agtcgttcgg tctggataaa 960 gccagcggcg cattgattgccaaaatcctt cccggcagcc ccgcagaacg tgccggcctg 1020 caggcgggcg acatcgtcctcagcctcgac ggcggagaaa tacgttcttc cggcgacctt 1080 cccgtcatgg tcggcgccattacgccggga aaagaagtca gcctcggcgt atggcgcaaa 1140 ggcgaagaaa tcacaatcaaagccaagctg ggcaacgccg ccgagcatac cggcgcatca 1200 tccaaaacag atgaagccccctacaccgaa cagcaatccg gtacgttctc ggtcgaatcc 1260 gcaggcatta cccttcagacacataccgac agcagcggca aacacctcgt cgtcgtacgg 1320 gtttccgacg cggcagaacgcgcaggctta aggcacggcg acgaaatcct agccgtcagg 1380 gcaagtcccc gtcaa 1395 4465 PRT Neisseria gonorrhoeae 4 Val Phe Lys Lys Tyr Gln Tyr Phe Ala LeuAla Ala Leu Cys Ala Ala 1 5 10 15 Leu Leu Ala Gly Cys Glu Lys Ala GlySer Phe Phe Gly Ala Asp Lys 20 25 30 Lys Glu Ala Ser Phe Val Glu Arg IleGlu His Thr Lys Asp Asp Gly 35 40 45 Ser Val Ser Met Leu Leu Pro Asp PheAla Gln Leu Val Gln Ser Glu 50 55 60 Gly Pro Ala Val Val Asn Ile Gln AlaAla Pro Ala Pro Arg Thr Gln 65 70 75 80 Asn Gly Ser Gly Asn Ala Glu ThrAsp Ser Asp Pro Leu Ala Asp Ser 85 90 95 Asp Pro Phe Tyr Glu Phe Phe LysArg Leu Val Pro Asn Met Pro Glu 100 105 110 Ile Pro Gln Glu Glu Ala AspAsp Gly Gly Leu Asn Phe Gly Ser Gly 115 120 125 Phe Ile Ile Ser Lys AsnGly Tyr Ile Leu Thr Asn Thr His Val Val 130 135 140 Ala Gly Met Gly SerIle Lys Val Leu Leu Asn Asp Lys Arg Glu Tyr 145 150 155 160 Thr Ala LysLeu Ile Gly Ser Asp Val Gln Ser Asp Val Ala Leu Leu 165 170 175 Lys IleAsp Ala Thr Glu Glu Leu Pro Val Val Lys Ile Gly Asn Pro 180 185 190 LysAsn Leu Lys Pro Gly Glu Trp Val Ala Ala Ile Gly Ala Pro Phe 195 200 205Gly Phe Asp Asn Ser Val Thr Ala Gly Ile Val Ser Ala Lys Gly Arg 210 215220 Ser Leu Pro Asn Glu Ser Tyr Thr Pro Phe Ile Gln Thr Asp Val Ala 225230 235 240 Ile Asn Pro Gly Asn Ser Gly Gly Pro Leu Phe Asn Leu Lys GlyGln 245 250 255 Val Val Gly Ile Asn Ser Gln Ile Tyr Ser Arg Ser Gly GlyPhe Met 260 265 270 Gly Ile Ser Phe Ala Ile Pro Ile Asp Val Ala Met AsnVal Ala Glu 275 280 285 Gln Leu Lys Asn Thr Gly Lys Val Gln Arg Gly GlnLeu Gly Val Ile 290 295 300 Ile Gln Glu Val Ser Tyr Gly Leu Ala Gln SerPhe Gly Leu Asp Lys 305 310 315 320 Ala Ser Gly Ala Leu Ile Ala Lys IleLeu Pro Gly Ser Pro Ala Glu 325 330 335 Arg Ala Gly Leu Gln Ala Gly AspIle Val Leu Ser Leu Asp Gly Gly 340 345 350 Glu Ile Arg Ser Ser Gly AspLeu Pro Val Met Val Gly Ala Ile Thr 355 360 365 Pro Gly Lys Glu Val SerLeu Gly Val Trp Arg Lys Gly Glu Glu Ile 370 375 380 Thr Ile Lys Ala LysLeu Gly Asn Ala Ala Glu His Thr Gly Ala Ser 385 390 395 400 Ser Lys ThrAsp Glu Ala Pro Tyr Thr Glu Gln Gln Ser Gly Thr Phe 405 410 415 Ser ValGlu Ser Ala Gly Ile Thr Leu Gln Thr His Thr Asp Ser Ser 420 425 430 GlyLys His Leu Val Val Val Arg Val Ser Asp Ala Ala Glu Arg Ala 435 440 445Gly Leu Arg His Gly Asp Glu Ile Leu Ala Val Arg Ala Ser Pro Arg 450 455460 Gln 465 5 1242 DNA Neisseria gonorrhoeae 5 atgctgctgc ccgactttgcccaactggtt caaagcgaag gcccggcagt cgtcaatatt 60 caggcagccc ccgccccgcgcacccaaaac ggcagcggca atgccgaaac cgattccgac 120 ccgcttgccg acagcgacccgttctacgaa tttttcaaac gcctcgtccc gaacatgccc 180 gaaatccccc aagaagaagcagatgacggc ggattgaact tcggttcggg cttcatcatc 240 agcaaaaacg gctacatcctgaccaatacc cacgtcgttg ccggtatggg cagtatcaaa 300 gtcctgctca acgacaagcgcgaatatacc gccaaactca tcggttcgga tgtccaatcc 360 gatgtcgccc ttctgaaaatcgacgcaacg gaagagctac ccgtcgtcaa aatcggcaat 420 cccaaaaatt tgaaaccgggcgaatgggtc gctgccatcg gcgcgccctt cggctttgac 480 aacagcgtga ccgccggcatcgtgtccgcc aaaggcagaa gcctgcccaa cgaaagctac 540 acacccttca tccaaaccgacgttgccatc aatccgggca attccggcgg cccgctgttc 600 aacttaaaag gacaggtcgtcggcatcaat tcgcaaatat acagccgcag cggcggattc 660 atgggcatct cctttgccatcccgattgac gttgccatga atgtcgccga acagctgaaa 720 aacaccggca aagtccaacgcggacaactg ggcgtgatta ttcaggaagt atcctacggt 780 ttggcacagt cgttcggtctggataaagcc agcggcgcat tgattgccaa aatccttccc 840 ggcagccccg cagaacgtgccggcctgcag gcgggcgaca tcgtcctcag cctcgacggc 900 ggagaaatac gttcttccggcgaccttccc gtcatggtcg gcgccattac gccgggaaaa 960 gaagtcagcc tcggcgtatggcgcaaaggc gaagaaatca caatcaaagc caagctgggc 1020 aacgccgccg agcataccggcgcatcatcc aaaacagatg aagcccccta caccgaacag 1080 caatccggta cgttctcggtcgaatccgca ggcattaccc ttcagacaca taccgacagc 1140 agcggcaaac acctcgtcgtcgtacgggtt tccgacgcgg cagaacgcgc aggcttaagg 1200 cacggcgacg aaatcctagccgtcagggca agtccccgtc aa 1242 6 414 PRT Neisseria gonorrhoeae 6 Met LeuLeu Pro Asp Phe Ala Gln Leu Val Gln Ser Glu Gly Pro Ala 1 5 10 15 ValVal Asn Ile Gln Ala Ala Pro Ala Pro Arg Thr Gln Asn Gly Ser 20 25 30 GlyAsn Ala Glu Thr Asp Ser Asp Pro Leu Ala Asp Ser Asp Pro Phe 35 40 45 TyrGlu Phe Phe Lys Arg Leu Val Pro Asn Met Pro Glu Ile Pro Gln 50 55 60 GluGlu Ala Asp Asp Gly Gly Leu Asn Phe Gly Ser Gly Phe Ile Ile 65 70 75 80Ser Lys Asn Gly Tyr Ile Leu Thr Asn Thr His Val Val Ala Gly Met 85 90 95Gly Ser Ile Lys Val Leu Leu Asn Asp Lys Arg Glu Tyr Thr Ala Lys 100 105110 Leu Ile Gly Ser Asp Val Gln Ser Asp Val Ala Leu Leu Lys Ile Asp 115120 125 Ala Thr Glu Glu Leu Pro Val Val Lys Ile Gly Asn Pro Lys Asn Leu130 135 140 Lys Pro Gly Glu Trp Val Ala Ala Ile Gly Ala Pro Phe Gly PheAsp 145 150 155 160 Asn Ser Val Thr Ala Gly Ile Val Ser Ala Lys Gly ArgSer Leu Pro 165 170 175 Asn Glu Ser Tyr Thr Pro Phe Ile Gln Thr Asp ValAla Ile Asn Pro 180 185 190 Gly Asn Ser Gly Gly Pro Leu Phe Asn Leu LysGly Gln Val Val Gly 195 200 205 Ile Asn Ser Gln Ile Tyr Ser Arg Ser GlyGly Phe Met Gly Ile Ser 210 215 220 Phe Ala Ile Pro Ile Asp Val Ala MetAsn Val Ala Glu Gln Leu Lys 225 230 235 240 Asn Thr Gly Lys Val Gln ArgGly Gln Leu Gly Val Ile Ile Gln Glu 245 250 255 Val Ser Tyr Gly Leu AlaGln Ser Phe Gly Leu Asp Lys Ala Ser Gly 260 265 270 Ala Leu Ile Ala LysIle Leu Pro Gly Ser Pro Ala Glu Arg Ala Gly 275 280 285 Leu Gln Ala GlyAsp Ile Val Leu Ser Leu Asp Gly Gly Glu Ile Arg 290 295 300 Ser Ser GlyAsp Leu Pro Val Met Val Gly Ala Ile Thr Pro Gly Lys 305 310 315 320 GluVal Ser Leu Gly Val Trp Arg Lys Gly Glu Glu Ile Thr Ile Lys 325 330 335Ala Lys Leu Gly Asn Ala Ala Glu His Thr Gly Ala Ser Ser Lys Thr 340 345350 Asp Glu Ala Pro Tyr Thr Glu Gln Gln Ser Gly Thr Phe Ser Val Glu 355360 365 Ser Ala Gly Ile Thr Leu Gln Thr His Thr Asp Ser Ser Gly Lys His370 375 380 Leu Val Val Val Arg Val Ser Asp Ala Ala Glu Arg Ala Gly LeuArg 385 390 395 400 His Gly Asp Glu Ile Leu Ala Val Arg Ala Ser Pro ArgGln 405 410 7 51 PRT Neisseria gonorrhoeae 7 Val Phe Lys Lys Tyr Gln TyrPhe Ala Leu Ala Ala Leu Cys Ala Ala 1 5 10 15 Leu Leu Ala Gly Cys GluLys Ala Gly Ser Phe Phe Gly Ala Asp Lys 20 25 30 Lys Glu Ala Ser Phe ValGlu Arg Ile Glu His Thr Lys Asp Asp Gly 35 40 45 Ser Val Ser 50 8 153DNA Neisseria gonorrhoeae 8 gtgttcaaaa aataccaata cttcgctttg gcggcactgtgtgccgcctt gctggcaggc 60 tgcgaaaagg caggcagctt tttcggtgcg gacaaaaaagaagcatcctt cgtagaacgc 120 atcgaacaca ccaaagacga cggcagtgtc agt 153 9 10PRT Neisseria gonorrhoeae 9 Val Phe Lys Lys Tyr Gln Tyr Phe Ala Leu 1 510 10 11 PRT Neisseria gonorrhoeae 10 Glu Ile Leu Ala Val Arg Ala SerPro Arg Gln 1 5 10

What is claimed is:
 1. An isolated NGSP polypeptide, which is apolypeptide of Neisseria spp, with the proviso that the Neisseria spp.is not N. meningitidis, and has a molecular weight of about 40 kD toabout 55 kD as determined in SDS polyacrylamide gel electrophoresis. 2.The NGSP polypeptide of claim 1, which has a molecular weight of about44 to 53 kD.
 3. The NGSP polypeptide of claim 1, wherein the Neisseriaspp. is selected from the group consisting of Neisseria ovis, Neisserialacunata, Neisseria osloensis, Neisseria bovis, and Neisseriagonorrhoeae.
 4. The NGSP polypeptide of claim 3, which Neisseria spp. isN. gonorrhoeae.
 5. The NGSP polypeptide of claim 1, comprising asequence selected from the group consisting of SEQ ID NOs: 4, 6, and 7,a sequence substantially homologous thereto, and a fragment thereof. 6.The NGSP polypeptide of claim 1 or a peptide fragment thereof, whichspecifically binds an antibody that specifically binds to a proteinhaving the sequence selected from the group consisting of SEQ ID NOs: 4,6, and
 7. 7. A peptide fragment of the NGSP polypeptide of claim
 1. 8. Apeptide fragment of the NGSP polypeptide of claim
 5. 9. A peptidefragment of the NGSP polypeptide of claim
 6. 10. An antibody thatspecifically binds the NGSP polypeptide of claim 1 or a fragmentthereof.
 11. An antibody that specifically binds the NGSP polypeptide ofclaim 5 or a fragment thereof.
 12. An antibody that specifically bindsthe NGSP polypeptide of claim 6 or a fragment thereof.
 13. The antibodyof claim 10, 11, or 12 which is a cytotoxic antibody that mediatescomplement killing of Neisseria gonorrhoeae.
 14. An antigeniccomposition comprising the NGSP polypeptide of any of claims 1, 5, or 6and a pharmaceutically acceptable carrier or diluent.
 15. An antigeniccomposition comprising the peptide fragment of claim 7, 8, or 9 and apharmaceutically acceptable carrier or diluent.
 16. The antigeniccomposition of claim 14 additionally comprising one or more adjuvants orimmunostimulatory compounds.
 17. The antigenic composition of claim 15additionally comprising one or more adjuvants or immunostimulatorycompounds.
 18. The antigenic composition of claim 16 further comprisingone or more immunogens selected from the group consisting of lipids,lipooligosaccharides, proteins, attenuated organisms and inactivatedwhole cells of a pathogenic organism.
 19. The antigenic composition ofclaim 18 wherein the lipid is a phospholipid.
 20. The antigeniccomposition of claim 17 further comprising optionally one or moreimmunogens selected from the group consisting of lipids,lipooligosaccharides, proteins, attenuated organisms and inactivatedwhole cells.
 21. The antigenic composition of claim 20 wherein the lipidis a phospholipid.
 22. A pharmaceutical composition comprising the NGSPpolypeptide of any of claims 1, 5 or 6 and a pharmaceutically acceptablecarrier or diluent.
 23. A pharmaceutical composition comprising thepeptide fragment of claim 7, 8, or 9 and a pharmaceutically acceptablecarrier or diluent.
 24. The pharmaceutical composition of claim 22additionally comprising one or more adjuvants or immunostimulatorycompounds.
 25. The pharmaceutical composition of claim 23 additionallycomprising one or more adjuvants or immunostimulatory compounds.
 26. Thepharmaceutical composition of claim 24 further comprising optionally oneor more immunogens selected from the group consisting of lipids,lipooligosaccharides, proteins, attenuated organisms and inactivatedwhole cells.
 27. The pharmaceutical composition of claim 26 wherein thelipid is a phospholipid.
 28. The pharmaceutical composition of claim 25further comprising optionally one or more immunogens selected from thegroup consisting of lipids, phospholipids, lipooligosaccharides,proteins, attenuated organisms and inactivated whole cells.
 29. Thepharmaceutical composition of claim 28 wherein the lipid is aphospholipid.
 30. A pharmaceutical composition comprising the antibodiesof claim 10, 11, 12 or
 13. 31. An isolated DNA comprising a nucleotidesequence encoding the NGSP polypeptide of claim 1, 5 or 6 or fragmentthereof.
 32. An isolated DNA having the sequence of SEQ ID NOs: 1, 2, 5,or 8, a fragment thereof, or the complement thereof.
 33. An isolated DNAencoding an NGSP polypeptide which comprises a nucleotide sequence thathybridizes under high stringency conditions to the sequence of SEQ IDNOs:1, 2, 5 or 8, or the complement thereof.
 34. An isolated DNA whichcomprises a nucleotide sequence that hybridizes under high stringencyconditions to the sequence of SEQ ID NQs: 1, 2, 5 or 8, or thecomplement thereof.
 35. A pharmaceutical composition comprising theisolated DNA of any one of claims 37, 38, 39 or
 40. 36. A method ofproducing an immune response in an animal comprising immunizing theanimal with an effective amount of the NGSP polypeptide of any of claims1, 5 or
 6. 37. A method of producing an immune response in an animalcomprising immunizing the animal with an effective amount of the peptidefragment of claim 7, 8, or
 9. 38. Plasmid pTLZ-NgHtrA#2 obtainable fromE. coli JM1O9 (pTLZ-NgHtrA#2), as deposited with the ATCC and assignednumber PTA-470.
 39. An antagonist which inhibits the activity orexpression of the NGSP polypeptide of claim
 5. 40. A method foridentifying compounds which interact with and inhibit or activate anactivity of the NGSP polypeptide of claim 5 comprising: contacting acomposition comprising the polypeptide with the compound to be screenedunder conditions to permit interaction between the compound and thepolypeptide to assess the interaction of a compound such interactionbeing associated with a second component capable of providing adetectable signal in response to the interaction of the polypeptide withthe compound; and determining the presence or absence of a signalgenerated from the interaction of the compound with the polypeptide. 41.A method for assaying for an agent that interacts with NGSP polypeptidecomprising: a. contacting a cell expressing NGSP polypeptide with anagent labeled with a detectable marker for a time sufficient to allowthe agent to interact with the polypeptide; b. washing the cells; and c.detecting any marker associated with the cells, in which any cellassociated marker indicates that the agent interacts with the NGSP 15polypeptide and wherein any agent that interacts with NGSP indicatesthat the agent is useful as a diagnostic, prophylactic or therapeuticagent against Neisseria infection.